Use of insulin for the treatment of cartilaginous disorders

ABSTRACT

The present invention relates to methods for the treatment and repair of cartilage, including cartilage damaged by injury or cartilaginous disorders, including arthritis, comprising the administration of insulin and/or insulin variants. Optionally, the administration may be in combination with a cartilage agent (e.g., peptide growth factor, catabolism antagonist, osteo-, synovial, anti-inflammatory factor), in an extended- or sustained-release form. Alternatively, the method provides for the treatment and repair of cartilage damaged by injury or cartilaginous disorders comprising the administration of insulin and/or insulin in combination with standard surgical techniques. Alternatively, the method provides for the treatment and repair of cartilage damaged by injury or cartilaginous disorders comprising the administration of chondrocytes previously treated with an effective amount of insulin and/or insulin variant.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 09/815,229 filedMar. 22, 2001, now U.S. Pat. No. 6,689,747, which claims priority under35 USC 119(e) to provisional application No. 60/192,103 filed Mar. 24,2000; all of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates generally to the repair of cartilage andthe treatment of cartilaginous disorders.

BACKGROUND OF THE INVENTION

Cartilaginous disorders broadly describe a collection of diseasescharacterized by degeneration of or metabolic abnormalities in theconnective tissues which are manifested by pain, stiffness andlimitation of motion of the affected body parts. The origin of thesedisorders can be pathological or as a result of trauma or injury.

Osteoarthritis (OA), also known as osteoarthrosis or degenerative jointdisease, is the result of a series of localized degenerative processesthat affect the articular structure and result in pain and diminishedfunction. The incidence of OA increases with age, and evidence of OA canbe detected at least one joint in the majority of the population by age65. OA is often accompanied by a local inflammatory component that mayaccelerate joint destruction.

OA is characterized by disruption of the smooth articulating surface ofcartilage, with early loss of proteoglycans (PG) and collagens, followedby formation of clefts and fibrillation, and ultimately byfull-thickness loss of cartilage. Coincident with the cartilaginouschanges are alterations in periarticular bone. The subchondral bonethickens and is slowly exposed. Bony nodules or osteophytes also oftenform at the periphery of the cartilage surface and occasionally growover the adjacent eroded areas. OA symptoms include local pain at theaffected joints, especially after use. With disease progression,symptoms may progress to a continuous aching sensation, local discomfortand cosmetic alterations such as deformity of the affected joint.

In contrast to the localized nature of OA, rheumatoid arthritis (RA) isa systemic, inflammatory disease which likely begins in the synovium,the tissues surrounding the joint space. The prevalence of RA is about ⅙that of OA in the general population of the United States. RA is achronic autoimmune disorder characterized by symmetrical synovitis ofthe joint and typically affects small and large diarthrodial joints,leading to their progressive destruction. As the disease progresses, thesymptoms of RA may also include fever, weight loss, thinning of theskin, multiorgan involvement, scleritis, corneal ulcers, the formationof subcutaneous or subperiosteal nodules and premature death. While thecause of RA and OA are distinctly different, the cytokines and enzymesinvolved in cartilage destruction appear to be similar.

Because mature chondrocytes have little potential for replication, andsince recruitment of other cell types is limited by the avascular natureof cartilage, mature cartilage has limited ability to repair itself. Forthis reason, transplantation of cartilage tissue or isolatedchondrocytes into defective joints has been used therapeutically.However, tissue transplants from donors run the risk of graft rejectionas well as possible transmission of infectious diseases. Although theserisks can be minimized by using the patient's own tissue or cells, thisprocedure requires further surgery, creation of a new lesion in thepatient's cartilage, and expensive culturing and growing ofpatient-specific cells. Better healing is achieved if the subchondralbone is penetrated, either by injury/disease or surgically, because thepenetration into the vaculature allows recruitment and proliferation ofundifferentiated cells to effect repair. Unfortunately, the biochemicaland mechanical properties of this newly formed fibrocartilage differfrom those of normal hyaline cartilage, resulting in inadequate oraltered function. Fibrocartilage does not have the same durability andmay not adhere correctly to the surrounding hyaline cartilage. For thisreason, the newly synthesized fibrocartilage may be more prone tobreakdown and loss than the original articular hyaline cartilage tissue.

Peptide growth factors are very significant regulators of cartilagegrowth and cartilage cell (chondrocyte) behavior (i.e., differentiation,migration, division, and matrix synthesis or breakdown) F. S. Chen etal., Am J. Orthop. 26: 396-406 (1997). Growth factors that have beenpreviously proposed to stimulate cartilage repair include insulin-likegrowth factor (IGF-1), Osborn, J. Orthop. Res. 7: 35-42 (1989); Florini& Roberts, J. Gerontol. 35: 23-30 (1980); basic fibroblast growth factor(bFGF), Toolan et al., J. Biomec. Mat. Res. 41: 244-50 (1998); Sah etal., Arch. Biochem. Biophys. 308: 137-47 (1994); bone morphogeneticprotein (BMP), Sato & Urist, Clin. Orthop. Relat. Res. 183: 180-87(1984); Chin et al., Arthritis Rheum. 34: 314-24 (1991) and transforminggrowth factor beta (TGF-β), Hill & Logan, Prog. Growth Fac. Res. 4:45-68 (1992); Gueme et al., J. Cell Physiol. 158: 476-84 (1994); Van derKraan et al., Ann. Rheum. Dis. 51: 643-47 (1992). Treatment with peptidegrowth factors alone, or as part of an engineered device forimplantation, could in theory be used to promote in vivo repair ofdamaged cartilage or to promote expansion of cells ex vivo prior totransplantation. However, because of their relatively small size, growthfactors are rapidly absorbed and/or degraded, thus creating a greattherapeutic challenge in trying to make them available to cells in vivoin sufficient quantity and for sufficient duration.

The present invention proposes to overcome this limitation by deliveryof a growth factor with a vehicle, and/or as a slow-release formulation.The ideal delivery vehicle is biocompatible, resorbable, has theappropriate mechanical properties, and results in no harmful degradationproducts.

Another method of stimulating cartilage repair is to inhibit theactivity of molecules which induce cartilage destruction and/or inhibitmatrix synthesis. One such molecule is the cytokine IL-1α, which hasdetrimental effects on several tissues within the joint, including thegeneration of synovial inflammation and up-regulation of matrixmetalloproteinases and prostaglandin expression. V. Baragi, et al., J.Clin. Invest. 96: 2454-60 (1995); V. M. Baragi et al., OsteoarthritisCartilage 5: 275-82 (1997); C. H. Evans et al., J. Leukoc. Biol. 64:55-61 (1998); C. H Evans and P. D. Robbins, J. Rheumatol. 24: 2061-63(1997); R. Kang et al., Biochem. Soc. Trans. 25: 533-37 (1997); R. Kanget al., Osteoarthritis Cartilage 5: 139-43 (1997). One means ofantagonizing IL-1α is through treatment with soluble IL-1 receptorantagonist (IL-1ra), a naturally occurring protein that prevents IL-1from binding to its receptor, thereby inhibiting both direct andindirect effects of IL-1 on cartilage. Other cytokines, such as IL-1β,tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), IL-6 andIL-8 have been linked to increased activation of synovialfibroblast-like cells, chondrocytes and/or macrophages. The inhibitionof these cytokines may be of therapeutic benefit in preventinginflammation and cartilage destruction. In fact, molecules which inhibitTNF-α activity have been shown to have potent beneficial effects on thejoints of patients with rheumatoid arthritis.

Nitric oxide also likely plays a substantial role in destruction ofcartilage. [Ashok et al., Curr. Opin. Rheum. 10: 263-268 (1998)]. Unlikenormal joint tissue which does not produce NO unless stimulated withcytokines such as IL-1, synovial membranes or cartilage obtained fromarthritic joints spontaneously produce large amounts of nitric oxide forup to 3 days after removal from the joint. In addition, increasedconcentrations of nitrites are found in synovial fluid of arthriticpatients. In addition to its direct stimulation of cartilage catabolism,nitric oxide present in an inflamed joint would likely lead to increasedvasodilation and permeability, further release of cytokines such asTNF-α and IL-1 from leukocytes, and stimulation of angiogenesis.Evidence for a causative role of NO in arthritis, comes from animalmodels where inhibition of NO has been shown to prevent IL-1 mediatedcartilage destruction and chondrocyte death as well as progression ofosteoarthritis. Finally, several agents (such as auranofin,glucocorticoids, cyclosporins, tetracyclines, and at least somenonsteroidal anti-inflammatory drugs including aspirin) currently usedfor the treatment of human rheumatic diseases, have been shown to reduceNO production and/or activity.

Prior studies in diabetic (with altered serum insulin levels) orabnormal (hypophysectomized) animals suggests that insulin is requiredfor optimal production of sulfated mucopolysaccharides and collagen, twomajor components of connective tissue. As early as 1957, insulin wasshown to increase the otherwise abnormally low uptake of labeled sulfateinto the skin of diabetic rats Schiller and Dorfman, J. Biol. Chem. 227:625-632 (1957). Similarly, insulin increased the otherwise low level ofsulfate uptake in aortae from diabetic rats. Cohen, M. P. and Foglia, V.G. Proc. Soc. Exp. Biol. Med. 132: 376-378 (1969); Proc. Soc. Exp. Biol.Med. 133: 1275-1278 (1970); Proc. Soc. Exp. Biol. Med. 135: 113-115(1970). Subsequently, insulin was shown to stimulate uptake of sulfateinto cartilage, but the identity of the molecules into which the sulfatewas incorporated was not determined. Furthermore, the effect of insulinon endogenous matrix turnover (i.e. protein breakdown or retentionwithin the matrix) was not assessed. Salmon, W. D., Jr., and Daughaday,W H. Endocrinol. 82: 493-499 (1957); J. Posever et al., J. OrthopaedicRes. 13: 832-827 (1995). While insulin may have direct effects onconnective tissues, at least some data suggests that the defects inconnective tissue metabolism found in diabetic animals, which can be atleast partially reversed by systemic administration of insulin, could bedue to circulating factor(s) induced by insulin and not due to directeffects of insulin on connective tissues (Spanheimer, R. G., Matrix 12:101-107 (1992).

Insulin has also been found to stimulate the growth of mouse fibroblastcultures, Paul and Pearson, J. Endocrinol. 21: 287-294 (1960), cartilagecells from hypophysectomized rats (Salmon, W. D., Jr., J. Lab. Clin.Med. 56: 673-681 (1960), cells in bone cultures (Prasad, G. C. andRajan, K. T., Acta Orthop. Scand. 41: 44-56 (1970), as well as cells inmany other systems (Gey, G. O. and Thalhimer, W., J. Amer. Med. Assoc.82: 1609 (1924); Lieberman, I., and Ove, P. O., J. Biol. Chem. 234:2754-2758 (1959); Younger, L. R., King, J. and Steiner, O. F., CancerRes. 26: 1408-1413 (1966); Schwartz, A. G. and Amos, H., Nature 219:1366-1367 (1968). Most of these very early studies were performed onwhole animals, organs, or tissues. Hajek and Solursh, Gene Comp.Endocrin. 25: 432-446 (1975) were among the first to show a directeffect of insulin on chondrocytes, derived from chick embryo sternalcartilage, in serum-free cultures. The stimulation of growth andmucopolysaccharide synthesis by insulin in these cultures may not besurprising given that the hormone insulin increases amino acid uptake,promotes a positive nitrogen balance, and favors overall proteinsynthesis. Similarly the increase in proteoglycan synthesis stimulatedby insulin in rat tumor cells derived from a Swarm rat chondrosarcomawas accompanied by an increase in incorporation of radioisotope intototal protein and thus may reflect a general increase in proteinsynthesis (Stevens, R. L. and Hascall, V. C., JBC 256: 2053-2058 (1981).Finally, it should be understood that high levels of insulin (10 μg/mlfor the chick cultures, Hajek and Solursh, supra) were used in many ofthese studies. At such high concentrations insulin binds to, andactivates, the insulin-like growth factor (IGF)-1 receptor, thusmimicking the effects of IGF-1 itself. Thus, the observed physiologicaleffects could be the result of IGF-1 receptor signalling and merely theresult of insulin signalling. Finally, systemic administration ofinsulin had no effect in an inflammatory, polyarthritic model in rats inwhich only the number of swollen joints was monitored. Roszkowski-Sliz,W., Acta Physiol. Pol. XXIV, 371-376 (1973). Since inflammation canoccur in the absence of cartilage and bone destruction and vice versa inanimal models, Joosten et al., J. Immunol. 163: 5049-5055 (1999), howinsulin may have affected the underlying joint tissues in this study isnot clear.

More than thirty years ago, high doses of insulin were injectedsubcutaneously into 10 patients, 7 of whom had rheumatoid arthritis,with the goal of inducing a hypoglycemic crisis, increasingcorticosteroid levels, and thus altering adrenal gland activity (M.Ippolito et al., Reumatismo 20(5): 561-64 (1967). Insulin treatmentassociated with cortisone resulted in overall beneficial effects for thepatients including regain of appetite, an increase in body weight, andimprovement in pain. These effects may be due to indirect activities ofinsulin, for example the ability of insulin to increase plasmacorticosteroid levels Since the authors were most interested in theeffects of systemic insulin on the “diencephalohypophysial system”, theanabolic effects of insulin on cartilage were not examined orconsidered. Furthermore, given the avascular nature of cartilage and therapid clearance of insulin in vivo, it is unlikely that much if any ofthe subcutaneously-delivered insulin would have been available tochondrocytes within the joints of the insulin-treated individuals.

Unlike OA, bone loss is a common feature of RA. Japanese patentapplication JO 59-234,826, filed Nov. 7, 1984 also speculated on thepotential application of insulin for the treatment of rheumatoidarthritis based on its induction of a bone marker, i.e. alkalinephosphatase activity, in the osteoblastic (bone) cell line, MC3T3-E1.However, the effects of insulin on cartilage tissue itself was notexamined or considered.

As the population ages, and the incidence of arthritis increases, aneffective therapy to induce repair of cartilage, including cartilagedamaged as a result of injury and/or disease, is urgently needed.

SUMMARY OF THE INVENTION

The present invention concerns methods for the treatment, repair andprotection of cartilage damaged as a result of a cartilaginous disorder,including that which results from disease and/or injury. Morespecifically, the invention concerns a method for the treatment, repairand protection of cartilage comprising administering an effective amountof insulin or an insulin variant. More specifically, the method providesfor administration of insulin or insulin variant in a sustained- orextended-release form.

In a further embodiment, the present invention concerns a method for thetreatment of cartilage damaged as a result of a cartilaginous disordercomprising contacting said cartilage with an effective amount of insulinand/or an insulin variant. Optionally, the cartilage is articularcartilage, and is contained within a mammal and the amount administeredis a therapeutically effective amount. Optionally, the cartilaginousdisorder is osteoarthritis, rheumatoid arthritis or an in injury.

In a further embodiment, the present invention concerns a method for thetreatment of cartilage damaged by injury or preventing the initial orcontinued damage comprising contacting said cartilage with an effectiveamount of insulin or insulin variant. More specifically, the injurytreated is microdamage or blunt trauma, a chondral fracture, anosteochondral fracture, or damage to tendons, menisci, or ligaments.More specifically, the cartilage is contained within a mammal, includinghumans, and the amount administered is a therapeutically effectiveamount. In a specific aspect, the injury can be the result of excessivemechanical stress or other biomechanical instability resulting from asports injury or obesity. Alternatively, the present invention concernsa method of treating or facilitating the repair of bone fracturescomprising contacting the region of the bone injury with an effectiveamount of insulin or insulin variant.

In a further embodiment, the present invention concerns a method for thetreatment of cartilage damaged or preventing initial or continued damageby a cartilaginous disorder and/or injury comprising contacting saidcartilage with an effective amount of a composition further comprisinginsulin or insulin variant. Alternatively, the composition furthercomprises a carrier, excipient or stabilizer. Alternatively, thecartilage is present in a mammal and the amount administered is atherapeutically effective amount. Alternatively, the composition may beadministered via injection or infusion by intravenous, intraarterial,intraperitoneal, intramuscular, intralesional, intraarticular or topicaladministration to a mammal and the amount administered is atherapeutically effective amount. Alternatively, the composition isinjected directly into the afflicted cartilaginous region or joint.

In a further embodiment, the present invention concerns a method for thetreatment of cartilage damaged or preventing initial or continued damageby a cartilaginous disorder and/or injury comprising administrating atherapeutically effective amount of a sustained or extended-releasecomposition containing insulin or insulin variant. Alternatively, thecartilage is present in a mammal and the amount administered is atherapeutically effective amount. More specifically, the extended- orsustained-release composition contains insulin or insulin variantformulated in a microencapsulation, a semi-permeable membrane of solidhydrophobic polymers, a biodegradable polymer(s), or a dispersion (e.g.,suspension or emulsion). More specifically, the semi-permeable membraneof solid hydrophobic polymer is polylactic-co-glycolic acid (PLGA), andthe biodegradable polymer is cross-linked hyaluronic acid (HA).Alternatively, the extended- or sustained-release insulin or insulinvariant composition further comprises a water-soluble polyvalent metalsalt. More specifically, the polyvalent metal salt includes the saltformed from a metallic cation and an inorganic or organic acid.

In a further embodiment, the invention concerns a method for treatingcartilage damaged or preventing initial or continued damage as a resultof injury or a cartilaginous disorder comprising contacting thecartilage with an effective amount of insulin or insulin variant incombination with an effective amount of a cartilage agent. Optionally,the cartilage is present inside a mammal and the amount administered isa therapeutically effective amount.

In another embodiment, the invention concerns a method of maintaining,enhancing or promoting the growth of chondrocytes in serum-free cultureby contacting the chondrocytes with an effective amount of insulin orinsulin variant. Alternatively, the method concerns contacting thechondrocyte with an effective amount of insulin or insulin variant in asustained or extended-release formulation. Alternatively, the presentinvention concerns a method of stimulating the regeneration orpreventing the degradation of cartilage resulting from injury or acartilaginous disorder by transplantation of an effective amount ofchondrocytes previously treated with an effective amount of insulin orinsulin variant.

In a further embodiment, the present invention concerns a method for thetreatment of cartilage damaged or preventing initial or continued damageas a result of injury or a cartilaginous disorder comprising contactingsaid cartilage with an effective amount of insulin and/or an insulinvariant in combination with any standard cartilage surgical technique.The present invention may be administered prior, after and/orsimultaneous to the standard cartilage surgical technique. In a specificaspect, the standard surgical technique may be selected from thefollowing procedures: cartilage shaving, abrasion chondroplasty, laserrepair, debridement, chondroplasty, microfracture with or withoutsubchondral bone penetration, mosaicplasty, cartilage cell allografts,stem cell autografts, costal cartilage grafts, chemical stimulation,electrical stimulation, perichondral autografts, periosteal autografts,cartilage scaffolds, shell (osteoarticular) autografts or allografts, orosteotomy.

In a further embodiment, the invention concerns nucleic acid encodinginsulin and/or insulin variants, and vectors and recombinant host cellscomprising such nucleic acid.

In another embodiment, the present invention concerns a therapeutic kit,comprising insulin and/or an insulin variant and a carrier, excipientand/or stabilizer (e.g. a buffer) in suitable packaging. The kitpreferably contains instructions for using insulin to treat cartilagedamaged or to prevent initial or continued damage to cartilage as aresult of a cartilaginous disorder. Alternatively, the kit may containinstructions for using insulin to treat a cartilaginous disorder.

In a further embodiment, the invention concerns an article ofmanufacture, comprising:

a container;

an instruction on the container; and

a composition comprising an active agent contained within the container;

wherein the composition is effective for treating a cartilaginousdisorder, the instruction on the container indicates that thecomposition can be used to treat a cartilaginous disorder, and theactive agent in the composition is an agent stimulating the repairand/or preventing the degradation of cartilage. In a preferred aspect,the active agent is insulin or an insulin variant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect insulin has on matrix synthesis in primaryarticular chondrocytes (ACs). Insulin at various concentrations (0.1-100nM) in serum-free media induced primary chondrocytes from porcinearticular cartilage to increase proteoglycan synthesis relative to theuntreated control samples (−) and was able to overcome interleukin 1α(IL-1α)-induced inhibition of synthesis. Media was analyzed forproteoglycan content using the colorimetric DMMB assay. “*” indicates asignificant (p<0.05) difference from that of the corresponding control(− or +IL1α).

FIG. 2 shows insulin treatment over an extended period of time (6 days)at various concentrations (1-100 nM) in serum-free media resulted inincreased metabolism of primary articular chondrocytes as determined bya colorimetric assay which measures metabolic activity of cultured cellsbased on the ability of viable cells to cleave the yellow tetrazoliumsalt MTT to form purple formazan crystals.

FIG. 3 shows the anabolic effects of insulin on articular cartilageexplants. Insulin treatment of porcine articular cartilage explantsevery day for 3 days resulted in a decrease in basal and IL-1α-inducedmatrix breakdown, as determined by measuring the amount of proteoglycansin the media using the DMMB assay (top panel, FIG. 3A). The treatmentalso significantly increased matrix synthesis and partially overcame theinhibition of matrix synthesis induced by IL-1α (bottom panel, FIG. 3B).

FIG. 4 shows the effects of insulin on retention of cartilage matrixproteins. The inhibition of matrix breakdown and induction of matrixsynthesis by insulin (as shown above in FIG. 3) resulted in an increasein the amount of matrix remaining in the cartilage tissue at the end ofthe 3 day treatment. Insulin was also able to overcome the decrease incartilage matrix content induced by IL-1α (+vs. Ins+).

FIG. 5 shows that insulin and IGF-1 decreased nitric oxide release by ACexplants. Media was harvested from articular cartilage explants whichwere treated with insulin (0.5-100 nM), IGF-1 (1-100 ng/ml) or mediaalone (−) in the absence (top panel, 5A (−)) or presence (bottom panel,5B (+)) of IL-1α. The amount of nitrite in the media was measured using2,3-diaminonapthalene (DAN) which reacts with nitrite under acidicconditions to form 1-(H)-naphthotriazole, a fluorescent product.

FIG. 6 shows a pictorial diagram of the patella assay. Followingintra-articular injection of a given “protein” into mouse knee joints,patellae were harvested, labelled with ³⁵S-sulfate, fixed, decalcified,dissected away from the underlying bone, and counted. The amount ofradioactivity incorporated into the cartilage is an indication of theextent of matrix synthesis.

FIG. 7 shows the effect of insulin on mouse patellae. Patellae wereharvested from mice and incubated with either IL-1α or insulin (Ins) for24 hours. During the last three hours, patellae were labelled with³⁵S-sulfate. Patellae were then processed as described in FIG. 6. Inthis model, IL-1α decreased and insulin increased matrix synthesis inthis intact articular cartilage assay (upper panel, FIG. 7A—in vitrotreatment). For in vivo analyses, mice were injected in one knee withinsulin, and in the other knee with buffer alone (PBS+0.1% BSA) everyday for three days, and patellae were harvested and analyzed in thepatella assay. Shown (bottom panel, FIG. 7B) are two separateexperiments. In the first experiment (left side), mice were injectedwith 6 μl of a 10 mg/ml insulin stock solution daily, and patellae wereharvested 3 hours after the third injection. In the second one (rightside), mice were injected with 31 μl of 10 mg/ml stock solution daily,and patellae were harvested 24 hours after the third injection. Resultsare expressed as the amount of radioactive incorporation relative tocontrol (contralateral knee) so that anything over 1.0 indicates aninduction of synthesis in the insulin-injected knee.

FIG. 8 shows the spontaneous joint degeneration which occurs in Hartleyguinea pigs as they age. The degeneration in the femorotibial joint ofmale Hartley guinea pigs occurs in an age-dependent manner, as shown byhistological analysis of articular cartilage at various ages (6, 9 and12 months, left side FIG. 8A). The histopathologic changes were gradedas minimal, moderate or severe (right side, FIG. 8B). Minimal changesinclude focal chondrocyte degradation, disruption of the superficiallayer of cartilage, and decreased toluidine blue matrix staining. Mildlesions were similar, but covered up to one-third of the surface andoften affected part of the middle layer. Moderate changes covered mostof the surface and included most of the middle layer. Severe changes hadextensive deep layer degeneration, osteophytes, chondrocyte cloning,subchondral bone thickening, synovial hyperplasia and fibrosis. Eachasterisk (*) represents one knee joint. (Data adapted from Wei, L. etal., Arthrits Rheum. 40: 2075-2083 (1997) and Bendele, A., Lab. Anim.Sci. 39: 115-121 (1989).

FIG. 9 shows that insulin has an anabolic effect on normal and diseasedcartilage. Articular cartilage was harvested from male Hartley guineapigs and cultured as explants in serum-free media without (−) or withinsulin (Insulin) at 100 nM. Incorporation of ³⁵S-sulfate was used anindication of proteoglycan synthesis. Note that at all ages (e.g., 1-2months—FIG. 9A, 6 months—FIG. 9B, and 11 months—FIG. 9C) insulinsignificantly stimulated proteoglycan synthesis.

FIG. 10 shows that insulin decreased catabolism of guinea pig articularcartilage. Insulin (Ins) decreased the amount of matrix breakdown asdetermined by measuring the amount of proteoglycans released fromarticular cartilage explants from 11 month old male Hartley guinea pigs(left side “media”). After three days of treatment, cartilage explantswere digested overnight, and the amount of proteoglycans remaining inthe tissue was determined using the DMMB assay (right side “tissue”).The decrease in matrix breakdown (media), and the increase in matrixsynthesis (FIG. 9) resulted in an increase in the amount of matrixremaining in the tissue at the end of the 3 day treatment (tissue).

FIG. 11 shows the effect of insulin on cartilage from diabetic mice.Patellae from diabetic, hypo-insulinemic mice (streptozotocin-treated)were harvested, and matrix synthesis was measured after culturing (18hours) in the absence (−) or presence (+Ins) of 100 nM insulin (FIG. 6).Basal synthesis was lower in the diabetic (Diab−) than in the controlmice (Con−). However, treatment with insulin brought synthesis levels inthe cartilage from diabetic mice (Diab+Ins) to levels comparable to thatof the untreated control (Con−Ins). Furthermore, the extent of inductionin control and diabetic tissues was similar.

FIG. 12 shows the cumulative release of HI from PLGA microspheres.Formulation II (see example 7) of PLGA encapsulated microspheres wereplaced in 10 mM histidine, 10 mM NaCl, 0.02% polysorbate 20, 0.02% NaN₃,pH 7.2 and incubated at 37° C. The entire release medium was replaced ateach sampling interval and the resulting release samples analyzed forbioactive insulin in an insulin kinase receptor assay (KIRA).

FIGS. 13A-13B show the release of insulin from PLGA encapsulation insynovial fluid. PLGA-Ins was incubated in synovial fluid harvested fromrat knee joints, and samples were taken daily to test for insulinactivity in an insulin kinase receptor assay (KIRA). As a control,synovial fluid spiked with insulin at 500 nM (SF+I-500) just before KIRAwas also tested. Synovial fluid itself had no detectable insulinactivity (data not shown). FIG. 13A shows release from days 1-3, whereasFIG. 13B is an expanded graph of data from FIG. 13A with release on days2 and 3. Results are expressed as nM concentration of active insulin.

FIG. 14 shows the activity of insulin (by KIRA) in samples harvesteddaily from samples incubated at 37° C. and 5% CO₂ under various cultureconditions. Each day media was harvested and tested for insulin activityin the insulin kinase receptor assay (KIRA). The results are expressedas nM concentration of active insulin. In FIG. 14A, samples were frominsulin which was incubated in serum-free explant media at 37° C. and 5%CO₂. In FIG. 14B, the same insulin-media solution as in FIG. 14A wasadded to porcine articular cartilage explants. In FIG. 14C,PLGA-microencapsulated insulin (PLGA-Ins) was added to explants, and inFIG. 14D, this PLGA-Ins solution was diluted 1:10 and added to explants.

FIGS. 15A-F shows the effect of PLGA-Ins on cartilage explants. FIGS.15A-B examine the effect on matrix breakdown. PLGA-Ins was added once(at time 0) to explants in the absence (FIG. 15A −) or presence (FIG.15B +) of IL-1α (1 ng/ml), and the amount of proteoglycans in the media(i.e., released by the explants) was measured using the DMMB assay.Media was changed at 24 h, and 48 hr, without adding additionalPLGA-Ins. In contrast, samples treated with insulin (10 nM) (Ins10)received fresh insulin with the media change at each timepoint (0, 24 h,48 h). Results are expressed as proteoglycans (μg) released over 24hours. A single treatment with PLGA-Ins was able to decrease both basaland IL-1α-induced matrix breakdown. FIGS. 15C-D examines the effect ofPLGA-Ins on matrix synthesis. PLGA-Ins was added to explants in theabsence (top) or presence (bottom, +) of IL-1α (1 ng/ml) for 3 days.PLGA-Ins was added only at time 0, while insulin (10 nM) (Ins10) wasadded with the media change at each timepoint (0, 24 h, 48 h). Duringthe last 15 hours of treatment, explants were labelled with ³⁵S-sulfate,and labelled matrix proteins were precipitated and counted (cpm). Asshown here, PLGA-Ins induced matrix synthesis, and overcame IL-1αinduced inhibition of proteoglycan synthesis. FIGS. 15E-F examines theeffect on nitric oxide (NO) production. PLGA-Ins was added to explantsin the absence (top) or presence (bottom, +) of IL-1α (1 ng/ml) and theamount of nitrite (μM) in the media was determined. PLGA-Ins was addedonly at time 0, while insulin (10 nM) (Ins10) was added with the mediachange at each timepoint (0, 24 h, 48 h). A single treatment withPLGA-Ins decreased basal nitric oxide production, and inhibitedinduction of nitric oxide synthesis by IL-1α.

FIG. 16 shows the in vivo effect of PLGA-insulin. PLGA-Ins was injectedinto the articular space of the hind leg of mice. Buffer (PBS+0.1% BSA)was injected into the contralateral knee as a control (−). Results areexpressed as amount of radioactivity (cpm)/patella. Each line representsa single mouse.

FIGS. 17A and 17B are diagrams representing the primary structure ofinsulin. Shown are a comparative diagram of the primary structure ofselected vertebrate insulin (FIG. 17A), and the primary structure ofporcine pro-insulin (FIG. 17B). The data was adapted from Hadley, M. E.,Endrocrinology, Prentice Hall Inc., 1988.

FIG. 18 shows the human native sequence of the A-chain of nativesequence A-chain (FIG. 18A)(SEQ ID NO:1), B-chain (FIG. 18B)(SEQ IDNO:2), native sequence proinsulin (FIG. 18C)(SEQ ID NO:3) and C-chain(FIG. 18D)(SEQ ID NO:4).

FIGS. 19A and 19B show the effect on matrix synthesis on human articularcartilage of IGF, IGF mutant which does not bind to IGF BPs and insulin.Shown is that insulin (Ins), but not IGF-1 (IGF) induces cartilagematrix synthesis in articular cartilage. Tissues were treated with theindicated agents for 24 hours during which proteoglycan synthesis wasmeasured by labeling cartilage with ³⁵S-sulphate. FIG. 19A shows theresults on tissue removed from a 63 year old white male withdegenerative joint disease (DJD) which was treated with IGF-1 (100ng/ml), desIGF (100 ng/ml) or insulin (13.15 nM—equivalent molarconcentration as 100 ng/ml IGF-1). FIG. 19B shows the results on tissueremoved from a 70 year old female with osteoarthritis (OA) which wastreated with IGF-1 (80 ng/ml), desIGF (80 ng/ml), or insulin (10.54nM—equivalent molar concentration as 80 ng/ml IGF-1).

DETAILED DESCRIPTION OF THE INVENTION

Osteoarthris v. Rheumatoid Arthritis:

Rheumatoid arthritis (RA) is a systemic, autoimmune, degenerativedisease that causes symmetrical disruptions in the synovium of bothlarge and small diarthroidal joints alike. As the disease progresses,symptoms of RA may include fever, weight loss, thinning of the skin,multiorgan involvement, scleritis, corneal ulcers, the formation ofsubcutaneous or subperiosteal nodules and premature death. In contrastto OA, RA symptoms appear during youth, extra-articular manifestationscan affect any organ system, and joint destruction is symmetrical andoccurs in both large and small joints alike. Extra-articular symptomscan include vasculitis, atrophy of the skin and muscle, subcutaneousnodules, lymphadenopathy, splenomegaly, leukopaenia and chronic anaemia.Furthermore, RA is heterogeneous in nature with a variable diseaseexpression and is associated with the formation of serum rheumatoidfactor in 90% of patients sometime during the course of the illness.

Interestingly, patients with RA also have a hyperactive immune system.The great majority of people with RA have a genetic susceptibilityassociated with increased activation of class II majorhistocompatibility complex molecules on monocytes and macrophages. Thesehistocompatibility complex molecules are involved in the presentation ofantigen to activated T cells bearing receptors for these class IImolecules. The genetic predisposition to RA is supported by theprevalence of the highly conserved leukocyte antigen DR subtype Dw4,Dw14 and Dw15 in human patients with very severe disease.

The activated monocytes and macrophages, in interacting with theappropriate T cells stimulate a cascade of events including furtheractivation of additional monocytes and macrophages, T cells, B cells andendothelial cells. With the upregulation of adhesion molecules,additional mononuclear cells and polymorphonuclear cells are attractedto the inflamed joint. This influx stimulates secretion of additionalchemotactic cytokines, thereby enhancing the influx of inflammatorycells into the synovium and synovial fluid.

Osteoarthritis (OA) is a localized degenerative disease that affectsarticular cartilage and bone and results in pain and diminished jointfunction. OA may be classified into two types: primary and secondary.Primary OA refers to the spectrum of degenerative joint diseases forwhich no underlying etiology has been determined. Typically, the jointaffected by primary OA are the interphalangeal joints of the hands, thefirst carpometacarpal joints, the hips, the knees, the spine, and somejoints in the midfoot. Interestingly, it appears that large joints, suchas the ankles, elbows and shoulders tend to be spared in primary OA. Incontrast, secondary OA often occurs as a result of defined injury ortrauma. Secondary arthritis can also be found in individuals withmetabolic diseases such as hemochromatosis and alkaptonuria,developmental abnormalities such as developmental dysplasia of the hips(congenital dislocation of the hips) and limb-length discrepancies,obesity, inflammatory arthritides such as rheumatoid arthritis or gout,septic arthritis, and neuropathic arthritis.

OA is a progressive, degenerative disorder. The degradation associatedwith OA initially appears as fraying and fibrillation of the articularcartilage surface as proteoglycans are lost from the matrix. Withcontinued joint use, surface fibrillation progresses, defects penetratedeeper into the cartilage, and pieces of cartilage tissue are lost. Inaddition, bone underlying the cartilage (subchondral bone) thickens,and, as cartilage is lost, bone becomes slowly exposed. With asymmetriccartilage destruction, disfigurement can occur. Bony nodules, calledosteophytes, often form at the periphery of the cartilage surface andoccasionally grow over the adjacent eroded areas. If the surface ofthese bony outgrowths is permeated, vascular outgrowth may occur andcause the formation of tissue plugs containing fibrocartilage.

Since cartilage is avascular, damage which occurs to the cartilage layerbut does not penetrate to the subchondral bone, leaves the job of repairto the resident chondrocytes, which have little intrinsic potential forreplication. However, when the subchondral bone is penetrated, itsvascular supply allows a triphasic repair process to take place. Thesuboptimal cartilage which is synthesized in response to this type ofdamage, termed herein “fibrocartilage” because of its fibrous matrix,has suboptimal biochemical and mechanical properties, and is thussubject to further wear and destruction. In a diseased or damaged joint,increased release of metalloproteinases (MMPs) such as collagenases,gelatinases, stromelysins, aggrecanases, and other proteases, leads tofurther thinning and loss of cartilage. In vitro studies have shown thatcytokines such as IL-1α, IL-1β, TNF-α, PDGF, GM-CSF, IFN-γ, TGF-β, LIF,IL-2 and IL-6, IL-8 can alter the activity of synovial fibroblast-likecells, macrophage, T cells, and/or osteoclasts, suggesting that thesecytokines may regulate cartilage matrix turnover in vivo. As such, anyof these cytokines could amplify and perpetuate the destructive cycle ofjoint degeneration in vivo. In fact, inhibition of IL-1 or TNF-αactivity in arthritic animals and humans has been shown to be aneffective way in which to at least slow the progression of arthritis.While the initiating events in RA and OA are clearly different,subsequent cartilage and bone loss in these two degenerative disordersappears to involve many of the same cytokines and proteinases.

The mechanical properties of cartilage are determined by its biochemicalcomposition. While the collagen architecture contributes to the tensilestrength and stiffness of cartilage, the compressibility (or elasticity)is due to its proteoglycan component. In healthy articular cartilage,type II collagen predominates (comprising about 90-95%), however,smaller amounts of types V, VI, IX, and XI collagen are also present.Cartilage proteoglycans (PG) include hydrodynamically large, aggregatingPG, with covalently linked sulfated glycosaminoglycans, as well ashydrodynamically smaller nonaggregating PG such as decorin, biglycan andlumican.

Types of Injuries to Cartilage

Injuries to cartilage fall into three categories: (1) microdamage orblunt trauma, (2) chondral fractures, and (3) osteochondral fractures.

Microdamage to chondrocytes and cartilage matrix may be caused by asingle impact, through repetitive blunt trauma, or with continuous useof a biomechanically unstable joint. In fact, metabolic and biochemicalchanges such as those found in the early stages of degenerativearthritis can be replicated in animal models involving repetitiveloading of articular cartilage. Radin et al., Clin. Orthop. Relat. Res.131: 288-93 (1978). Such experiments, along with the distinct pattern ofcartilage loss found in arthritic joints, highlight the role thatbiomechanical loading plays in the loss of homeostasis and integrity ofarticular cartilage in disease. Radin et al., J Orthop Res. 2: 221-234(1984); Radin et al., Semin Arthritis Rheum (suppl. 2) 21: 12-21 (1991);Wei et al., Acta Orthop Scand 69: 351-357 (1998). While chondrocytes mayinitially be able to replenish cartilage matrix with proteoglycans at abasal rate, concurrent damage to the collagen network may increase therate of loss and result in irreversible degeneration. Buckwalter et al.,J. Am. Acad. Orthop. Surg. 2: 192-201 (1994).

Chondral fractures are characterized by disruption of the articularsurface without violation of the subchondral plate. Chondrocyte necrosisat the injury site occurs, followed by increased mitotic and metabolicactivity of the surviving chondrocytes bordering the injury which leadsto lining of the clefts of the articular surface with fibrous tissue.The increase in chondrocyte activity is transitory, and the repairresponse results in insufficient amount and quality of new matrixcomponents.

Osteochondral fractures, the most serious of the three types ofinjuries, are lesions crossing the tidemark into the underlyingsubchondral plate. In this type of injury, the presence of subchondralvasculature elicits the three-phase response typically encountered invascular tissues: (1) necrosis, (2) inflammation, and (3) repair.Initially the lesion fills with blood and clots. The resulting fibrinclot activates an inflammatory response and becomes vascularized repairtissue, and the various cellular components release growth factors andcytokines including transforming growth factor beta (TGF-beta),platelet-derived growth factor (PDGF), bone morphogenic proteins, andinsulin-like growth factors I and II. Buckwalter et al., J. Am. Acad.Orthop. Surg. 2: 191-201 (1994).

The initial repair response associated with osteochondral fractures ischaracterized by recruitment, proliferation and differentiation ofprecursors into chondrocytes. Mesenchymal stem cells are deposited inthe fibrin network, which eventually becomes a fibrocartilaginous zone.F. Shapiro et al., J. Bone Joint Surg. 75: 532-53 (1993); N. Mitchelland N. Shepard, J. Bone Joint Surg. 58: 230-33 (1976). These stem cells,which are believed to come from the underlying bone marrow rather thanthe adjacent articular surface, progressively differentiate intochondrocytes. At six to eight weeks after injury, the repair tissuecontains chondrocyte-like cells in a matrix of proteoglycans andpredominantly type II collagen, with some type I collagen. T. Furukawaet al., J. Bone Joint Surg. 62: 79-89 (1980); J. Cheung et al.,Arthritis Rheum. 23: 211-19 (1980); S. O. Hjertquist & R. Lemperg, Calc.Tissue Res. 8: 54-72 (1971). However, this newly deposited matrixdegenerates, and the chondroid tissue is replaced by more fibrous tissueand fibrocartilage and a shift in the synthesis of collagen from type IIto type I. H. S. Cheung et al., J. Bone Joint Surg. 60: 1076-81 (1978);D. Hamerman, “Prospects for medical intervention in cartilage repair,”Joint cartilage degradation: Basic and clinical aspects, Eds. Woessner JF et al., (1993); Shapiro et al., J. Bone Joint Surg. 75: 532-53 (1993);N. Mitchell & N. Shepard, J. Bone Joint Surg. 58: 230-33 (1976); S. O.Hjertquist & R. Lemperg, Calc. Tissue Res. 8: 54-72 (1971). Earlydegenerative changes include surface fibrillation, depletion ofproteoglycans, chondrocyte cloning and death, and vertical fissuringfrom the superficial to deep layers. At one year post-injury, the repairtissue is a mixture of fibrocartilage and hyaline cartilage, with asubstantial amount of type I collagen, which is not found in appreciableamounts in normal articular cartilage. T. Furukawa, et al., J. BoneJoint Surg. 62: 79-89 (1980).

From a clinical viewpoint, the fibrocartilaginous repair tissue mayfunction satisfactorily for a certain length of time. However,fibrocartilage has inferior biomechanical properties relative to that ofnormal hyaline cartilage. Collagen fibers are arrayed in a randomorientation with a lower elastic modulus in fibrocartilage than innormal hyaline cartilage. J. Colletti et al., J. Bone Joint Surg. 54:147-60 (1972). The permeability of the repair tissue is also elevated,thus reducing the fluid-pressure load-carrying capacity of the tissue.H. Mankin et al., “Form and Function of Articular Cartilage”,Orthopaedic Basic Science, Ed: Simon & Schuster, American Academy ofOrthopeadic Surgeons, Rosemont, Ill. (1994). These changes result inincreased viscoelastic deformation, making the repair tissue less ableto withstand repetitive loading than normal articular cartilage.Glycosaminoglycan (GAG) levels in the cartilage adjacent toosteochondral defects have been reported to be reduced by 42% of normalvalues, indicating that injury leads to degeneration beyond the initialdefect. Osteoarthritis Cartilage 3: 61-70 (1995).

Chondrocyte Transplantation and Survival

The transplantation of chondrocytes, the cells responsible for secretingcartilage matrix, has also been suggested as a means of effectingcartilage repair. However, the disadvantages of allografts e.g. thepossibility of the host's immunogenic response as well as thetransmission of viral and other infectious diseases, has effectivelylimited the scope of allogenic chondrocyte transplantation. Althoughthese risks can be minimized by using the patient's own tissue or cells,this procedure requires further surgery, creation of a new lesion in thepatient's cartilage, and expensive culturing and growing ofpatient-specific cells.

When cultured as monolayers on tissue culture dishes, isolatedchondrocytes will de-differentiate, and with time in culture, come toresemble fibroblasts. For example, collagen production will switch frompredominantly type II to type I, and cells will synthesize an increasedproportion of hyaluronic acid relative to the total glycosaminoglycan(GAG) content. W. Green, Clin. Orthop. Relat. Res. 124: 237-50 (1977).However, chondrocytes grown in collagen gels or as aggregate cultureswill maintain normal morphology, proteoglycan and type II collagensynthesis as well as and retain their ability to accumulatemetachromatic matrix in vitro. Thus, under these conditions, chondocyteswill remain relatively differentiated and phenotypically stable for upto several weeks in vitro. T. Kimura et al., Clin. Orthop. Relat. Res.186: 231-39 (1984).

Tissue Engineering:

The difficulties and expense associated with the culturing ofchondrocytes has led to the design of chondrocyte-seeded or cell-freeimplants for articular cartilage repair using a variety of biomaterials,including: demineralized or enzymatically treated bone, L. Dahlberg. etal., J. Orthop. Res. 9: 11-19 (1991); B. C. Toolan et al., J. Biotned.Mat. Res. 41: 244-50 (1998); polylactic acid C. R., Chu et al., J.Biomed. Mat. Res. 29: 1147-54 (1995); polyglycolic acid C. A. Vacanti etal., Mat. Res. Soc. Symp. Proc. 252: 367-74 (1992);hydroxyapaptite/Dacron composites, K. Messner & J. Giliquist,Biomaterials 14: 513-21 (1993); fibrin, D. A. Hendrickson et al., J.Orthop. Res. 12: 485-97 (1994); collagen gels, D. Grande et al., J.Orthop. Res. 7: 208-18 (1989), S. Wakitani et al., J. Bone Joint Surg.71: 74-80 (1989), S. Wakitani et al., J. Bone Joint Surg. 76: 579-92(1994); and collagen fibers, J. M. Pachence et al., “Development of atissue analog for cartilage repair,” Tissue inducing biomaterials, Eds,L. Cima & E. Ron, Materials Research Soc. Press., Pittsburgh, Pa.(1992); B. C. Toolan et al., J. Biomed. Mat. Res. 31: 273-80 (1996).Alternative tissues employed include synovial tissue, A. G. Rothwell,Orthopedics 13: 433-42 (1990); or tissues rich in mesenchymal stem cells(e.g., bone marrow or periosteal tissue), K. Messner & J. Gillquist,Mat. Res. Soc. Symp. Proc. 252: 367-74 (1992).

Standard Cartilage Surgical Techniques:

The present method may also be administered in combination with anystandard cartilage surgical technique. Standard surgical techniques aresurgical procedures which are commonly employed for therapeuticmanipulations of cartilage, including: cartilage shaving, abrasionchondroplasty, laser repair, debridement, chondroplasty, microfracturewith or without subchondral bone penetration, mosaicplasty, cartilagecell allografts, stem cell autografts, costal cartilage grafts, chemicalstimulation, electrical stimulation, perichondral autografts, periostealautografts, cartilage scaffolds, shell (osteoarticular) autografts orallografts, or osteotomy. These techniques are described and discussedin greater detail in Frenkel et al., Front. Bioscience 4: d671-685(1999).

Cartilage Agents:

In combination with or in lieu of tissue engineering, the administrationof cartilage agents (e.g., peptide growth factors) has been consideredas a way to augment cartilage repair. Cartilage agents are verysignificant regulators of cartilage cell differentiation, migration,adhesion, and metabolism. F. S. Chen et al., Am J. Orthop. 26: 396-406(1997). Because cartilage agents are soluble proteins of relative smallmolecular mass and are rapidly absorbed and/or degraded, a greatchallenge exists in making them available to cells in sufficientquantity and for sufficient duration. Secreted proteins may thus need tobe incorporated into engineered, implantable devices for maximumeffectiveness. The ideal delivery vehicle is biocompatible, resorbable,has the appropriate mechanical properties, and degrades into non-toxicby-products.

Several secreted peptides have the potential to induce host cartilagerepair without transplantation of cells. Insulin-like growth factor(IGF-1) stimulates both matrix synthesis and cell proliferation inculture, K. Osborn. J. Orthop. Res. 7: 35-42 (1989), and insufficiencyof IGF-1 may have an etiologic role in the development ofosteoarthritis. R. D. Coutts, et al., Instructional Course Lect. 47:487-94, Amer. Acad. Orthop. Surg. Rosemont, Ill. (1997). Some studiesindicate that serum IGF-1 concentrations are lower in osteoarthriticpatients than control groups, while other studies have found nodifference. Nevertheless, both serum IGF-1 levels and chondrocyteresponsiveness to IGF-1 decrease with age. J. R. Florini & S. B.Roberts, J. Gerontol. 35: 23-30 (1980). Thus, both the decreasedavailability of IGF-1 as well as diminished chondrocyte responsivenessto IGF-1 may contribute to cartilage homeostasis and lead todegeneration with advancing age.

IGF-1 has been proposed for the treatment of prevention ofosteoarthritis. In fact, intra-articular administration of IGF-1 incombination with sodium pentosan polysulfate (a chondrocyte catabolicactivity inhibitor) caused improved histological appearance, andnear-normal levels of degradative enzymes (neutral metalloproteinasesand collagenase), tissue inhibitors of metalloproteinase and matrixcollagen. R. A. Rogachefsky, et al., Ann. NY Acad. Sci. 732: 889-95(1994). The use of IGF-1 either alone or as an adjuvant with othergrowth factors to stimulate cartilage regeneration has been described inWO 91/19510, WO 92/13565, U.S. Pat. No. 5,444,047, EP 434,652,

Bone morphogenetic proteins (BMPs) are members of the large transforminggrowth factor beta (TGF-β) family of growth factors. In vitro and invivo studies have shown that BMP induces the differentiation ofmesenchymal cells into chondrocytes. K. Sato & M. Urist, Clin. Orthop.Relat. Res. 183: 180-87 (1984). Furthermore, skeletal growth factor andcartilage-derived growth factors have synergistic effects with BMP, asthe combination of these growth factors with BMP and growth hormoneinitiates mesenchymal cell differentiation. Subsequent proliferation ofthe differentiated cells are stimulated by other factors. D. J. Hill & ALogan, Prog. Growth Fac. Res. 4: 45-68 (1992).

Transforming growth factor beta (TGF-β) is produced by osteoblasts,chondrocytes, platelets, activated lymphocytes, and other cells. R. D.Coutts et al., supra. TGF-β can have both stimulatory and inhibitoryproperties on matrix synthesis and cell proliferation depending on thetarget cell, dosage, and cell culture conditions. P. Guerne et al., J.Cell Physiol. 158: 476-84 (1994); H. Van Beuningen et al., Ann. Rheum.Dis. 52: 185-91 (1993); P. Van der Kraan et al., Ann. Rheum. Dis. 51:643-47 (1992). Furthermore, as with IGF-1, TGF-β, responsiveness isdecreased with age. P. Guerne et al., J. Cell Physiol. 158: 476-84(1994). However, TGF-β is a more potent stimulator of chondrocyteproliferation than other growth factors, including platelet-derivedgrowth factor (PDGF), bFGF, and IGF-1 (Guerne et al., supra), and canstimulate proteoglycan production by chondrocytes. TGF-β alsodown-regulates the effects of cytokines which stimulate chondrocytecatabolism Van der Kraan et al., supra. In vivo, TGF-β inducesproliferation and differentiation of mesenchymal cells into chondrocytesand enhances repair of partial-thickness defects in rabbit articularcartilage. E. B. Hunziker & L. Rosenberg, Trans. Orthopaed. Res. Soc.19: 236 (1994).

Antagonism of Cartilage Catabolism

Cartilage matrix degradation is believed to be due to cleavage of matrixmolecules (proteoglycans and collagens) by proteases (reviewed inWoessner J F Jr., “Proteases of the extracellular matrix”, in Mow, V.,Ratcliffe, A. (eds): Structure and Function of Articular Cartilage. BocaRaton, Fla., CRC Press, 1994 and Smith R. L., Front. In Biosci.4:d704-712. While the key enzymes involved in matrix breakdown have notyet been clearly identified, matrix metalloproteinases (MMPs) and“aggrecanases” appear to play key roles in joint destruction. Inaddition, members of the serine and cysteine family of proteinases (forexample the cathepsins and urokinase or tissue plasminogen activator(uPA and tPA)) may also be involved. Plasmin, urokinase plasminogenactivator (uPA) and tissue plasminogen activator (tPA may play animportant role in the activation pathway of the metalloproteinases.Evidence connects the closely related group of cathepsin B, L and S tomatrix breakdown, and these cathepsins are somewhat increased in OA.Many cytokines, including IL-1, TNF-α and LIF induce MMP expression inchondrocytes. Induction of MMPs can be antagonized by TGF-β and 1L-4 andpotentiated, at least in rabbits, by FGF and PDGF. As shown by animalstudies, inhibitors of these proteases (MMPs and aggrecanases) may atleast partially protect joint tissue from damage in vivo.

Other methods of stimulating cartilage repair include blocking theeffects of molecules which are associated with cartilage destruction.For example, both IL-1 (α and β) and nitric oxide are substances withknown catabolic effects on cartilage. The cytokine IL-1 causes cartilagebreakdown, including the generation of synovial inflammation andup-regulation of matrix metalloproteinases and aggrecanases. V. Baragi,et al., J. Clin. Invest. 96: 2454-60 (1995); V. M. Baragi et al.,Osteoarthritis Cartilage 5: 275-82 (1997); C. H. Evans et al., J.Leukoc. Biol. 64: 55-61 (1998); C. H Evans and P. D. Robbins, J.Rheumatol. 24: 2061-63 (1997); R. Kang et al., Biochem. Soc. Trans. 25:533-37 (1997); R. Kang et al., Osteoarthritis Cartilage 5: 13943 (1997).Because high levels of IL-1 are found in diseased joints and IL-1 isbelieved to play a pivotal role in initiation and development ofarthritis, inhibition of IL-1 activity may prove to be a successfultherapy. In mammals only one protease, named interleukin 1β-convertase(ICE), can specifically generate mature, active IL-1β. Inhibition of ICEhas been shown to block IL-1β production and may slow arthriticdegeneration (reviewed in Martel-Pelletier J. et al. Front. Biosci. 4:d694-703). The soluble IL-1 receptor antagonist (IL-1ra), a naturallyoccurring protein that can inhibit the effects of IL-1 by preventingIL-1 from interacting with chondrocytes, has also been shown to beeffective in animal models of arthritis and is currently being tested inhumans for its ability to prevent the incidence or progression ofarthritis.

Nitric oxide (NO) plays a substantial role in the destruction ofcartilage. Ashok et al., Curr. Opin. Rheum. 10: 263-268 (1998). Unlikenormal cartilage which does not produce NO unless stimulated withcytokines such as IL-1α, cartilage obtained from osteoarthritic jointsproduces large amounts of nitric oxide for over 3 days in culturedespite the absence of added stimuli. Moreover, inhibition of NOproduction has been shown to prevent IL-1α mediated cartilagedestruction and chondrocyte death as well as progression ofosteoarthritis in animal models. Thus, inhibition of NO may be one wayto prevent cartilage destruction.

As with IL1α and β, TNF-α is synthesized by chondrocytes, induces matrixbreakdown, inhibits matrix synthesis, and is found at high levels inarthritic joints. TNF-α also synergizes with IL-1 in terms of cartilagedestruction. Inhibition of TNF-α activity, in arthritic animals andhumans has been shown to inhibit progression of arthritis.

Leukemia inhibitory factor (LIF), which is synthesized by both cartilageand synovium, is present in human synovial fluids. Because LIF inducesthe synthesis of matrix metalloproteinases (MMPs) by chondrocyte, it maybe involved in the breakdown of the cartilaginous matrix.

Interferon-gamma (IFN-γ) inhibits proteoglycan synthesis by humanchondrocytes without enhancing its breakdown. Indeed, IFN-γ may suppressproteoglycan loss by inhibiting the induction of MMPs.

Interleukin 8, a potent chemotactic cytokine for polymorphonuclearneutrophils (PMN), is synthesized by a variety of cells includingmonocytes/macrophages, chondrocytes and fibroblasts and is induced byTNF-α. In OA patients, IL-β, IL-6, TNF-α and IL-8 are all found in thesynovial fluid. IL-8 can enhance the release of inflammatory cytokinesin human mononuclear cells, including that of IL-1β, IL-6 and TNF-α,which may further modulate the inflammatory reaction (reviewed inMartel-Pelletier J. et al., Front. Biosci. 4: d694-703).

IL-6 has also been proposed as a contributor to the OA pathologicalprocess by increasing inflammatory cells in the synovial tissue and bystimulating the proliferation of chondrocytes. In addition, IL-6 canamplify the effects of IL-1 on MMP synthesis and inhibition ofproteoglycan production (reviewed in Martel-Pelletier J. et al. Front.Biosci. 4: d694-703).

Interleukin 17 upregulates production of IL-1β, TNF-α, IL-6 and MMPs inhuman macrophages. IL-17 also induces NO production in chondrocytes, andis expressed in arthritic, but not normal joints (reviewed inMartel-Pelletier J. et al. Front. Biosci. 4: d694-703).

Basic fibroblast growth factor (bFGF), which is synthesized bychondrocytes, can induce articular chondrocyte replication. B. C. Toolanet al., J. Biomed. Mat. Res. 41: 244-50 (1998). In explants taken fromyoung animals, bFGF in small amounts (e.g., 3 ng/ml) stimulatessynthesis and inhibits breakdown of proteoglycans, while higher levels(e.g., 30-300 ng/ml) has exactly the opposite effect (i.e., synthesisinhibition and enhanced breakdown). In adult tissues, higher doses ofFGF stimulated proteoglycan, protein and collagen synthesis with no cellproliferation. R. L. Sah et al., Arch. Biochem. Biophys. 308: 137-47(1994). bFGF also regulates cartilage homeostasis by inducing theautocrine release from chondrocytes of interleukin 1 (IL-1), a potentstimulator of catabolic behavior in cartilage. bFGF further enhancesIL-1-mediated protease release, perhaps through its ability toupregulate IL-1 receptors on chondrocytes. J. E. Chin et al., ArthritisRheum. 34: 314-24 (1991)]. Similarly, platelet-derived growth factor(PDGF) can potentiate the catabolic effects of IL-1 and presumably ofTNF-α. However, some evidence suggests that in human cartilage bFGF andPDGF may have an anticatabolic effect; whether this phenomenon isspecies-specific or an effect of age remains to be determined.

While inflammation does not appear to be the initiating even inosteoarthritis, inflammation does occur in osteoarthritic joints. Theinflammatory cells (i.e. monocytes, macrophages, and neutrophils) whichinvade the synovial lining after injury and during inflammation producemetalloproteinases as well as catabolic cyokines which can contribute tofurther release of degradative enzymes. Although inflammation and jointdestruction do not show perfect correlation in all animal models ofarthritis, agents such as IL-4, IL-10 and IL-13 which inhibitinflammation also decrease cartilage and bone pathology in arthriticanimals (reviewed in Martel-Pelletier J. et al. Front. Biosci. 4:d694-703). Application of agents which inhibit inflammatory cytokinesmay slow OA progression by countering the local synovitis which occursin OA patients.

Numerous studies show that members of the tetracycline family ofantibiotics are effective in inhibiting collagenase and gelatinaseactivity. Oral administration of one of these, doxycycline, proved todecrease both collagenase and gelatinase activity in cartilage fromendstage hip osteoarthritis. These data suggest that an effective oraldose of doxycycline may slow down progression of osteoarthritis. SmithR. L. Front. Biosci. 4: d704-712.

OA involves not only the degeneration of articular cartilage leading toeburnation of bone, but also extensive remodelling of subchondral boneresulting in the so-called sclerosis of this tissue. These bony changesare often accompanied by the formation of subchondral cysts as a resultof focal resorption. Agents which inhibit bone resorption, i.e.osteoprotegerin or bisphosphonates have shown promising results inanimal model of arthritis. Kong et al. Nature 402: 304-308.

I. Definitions

The term “cartilaginous disorder” refers to a collection of diseaseswhich are further manifested by symptoms of pain, stiffness and/orlimitation of motion of the affected body parts, including that whichresults for disease or injury. Included within the scope of“cartilaginous disorders” is “degenerative cartilaginous disorders”—acollection of disorders characterized, at least in part, by degenerationor metabolic derangement of connective tissues of the body, includingnot only the joints or related structures, including muscles, bursae(synovial membrane), tendons and fibrous tissue, but also the growthplate. In one embodiment, the term includes “articular cartilagedisorders” which are characterized by disruption of the smooth articularcartilage surface and degradation of the cartilage matrix. Additionalpathologies include nitric oxide production, and inhibition or reductionof matrix synthesis.

Included within the scope of “articular cartilage disorder” areosteoarthritis (OA) and rheumatoid arthritis (RA). OA defines not asingle disorder, but the final common pathway of joint destructionresulting from multiple processes. OA is characterized by localizedasymmetric destruction of the cartilage commensurate with palpable bonyenlargements at the joint margins. OA typically affects theinterphalangeal joints of the hands, the first carpometacarpal joint,the hips, the knees, the spine, and some joints in the midfoot, whilelarge joints, such as the ankles, elbows and shoulders tend to bespared. OA can be associated with metabolic diseases such ashemochromatosis and alkaptonuria, developmental abnormalities such asdevelopmental dysplasia of the hips (congenital dislocation of thehips), limb-length discrepancies, including trauma and inflammatoryarthritides such as gout, septic arthritis, neuropathic arthritis. OAmay also develop after extended biomechanical instability, such as thatresulting from sports injury or obesity.

Rheumatoid arthritis (RA) is a systemic, chronic, autoimmune disordercharacterized by symmetrical synovitis of the joint and typicallyaffects small and large diarthroid joints alike. As RA progresses,symptoms may include fever, weight loss, thinning of the skin,multiorgan involvement, scieritis, corneal ulcers, the formation ofsubcutaneous or subperiosteal nodules and even premature death. Thesymptoms of RA often appear during youth and can include vasculitis,atrophy of the skin and muscle, subcutaneous nodules, lymphadenopathy,splenomegaly, leukopaenia and chronic anaemia.

Furthermore, the term “degenerative cartilaginous disorder” may includesystemic lupus erythematosus and gout, amyloidosis or Felty's syndrome.Additionally, the term covers the cartilage degradation and destructionassociated with psoriatic arthritis, osteoarthrosis, acute inflammation(e.g., yersinia arthritis, pyrophosphate arthritis, gout arthritis(arthritis urica), septic arthritis), arthritis associated with trauma,inflammatory bowel disease (e.g., Crohn's disease, ulcerative colitis,regional enteritis, distal ileitis, granulomatous enteritis, regionalileitis, terminal ileitis), multiple sclerosis, diabetes (e.g.,insulin-dependent and non-insulin dependent), obesity, giant cellarthritis and Sjögren's syndrome.

Examples of other immune and inflammatory diseases, at least some ofwhich may be treatable by the methods of the invention include, juvenilechronic arthritis, spondyloarthropathies, systemic sclerosis(scleroderma), idiopathic inflammatory myopathies (dermatomyositis,polymyositis), systemic vasculitis, sarcoidosis, autoimmune hemolyticanemia (immune pancytopenia, paroxysmal nocturnal hemoglobinuria),autoimmune thrombocytopenia (idiopathic thrombocytopenic purpura,immune-mediated thrombocytopenia), thyroiditis (Grave's disease,Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophicthyroiditis) autoimmune inflammatory diseases (e.g., allergicencephalomyelitis, multiple sclerosis, insulin-dependent diabetesmellitus, autoimmune uveoretinitis, thyrotoxicosis, autoimmune thyroiddisease, pernicious anemia) and allograft rejection, diabetes mellitus,immune-mediated renal disease (glomerulonephritis, tubulointerstitialnephritis), demyelinating diseases of the central and peripheral nervoussystems such as multiple sclerosis, idiopathic demyelinatingpolyneuropathy or Guillain-Barré syndrome, and chronic inflammatorydemyelinating polyneuropathy, hepatobiliary diseases such as infectioushepatitis (hepatitis A, B, C, D, E and other non-hepatotropic viruses),autoimmune chronic active hepatitis, primary biliary cirrhosis,granulomatous hepatitis, and sclerosing cholangitis, gluten-sensitiveenteropathy, and Whipple's disease, autoimmune or immune-mediated skindiseases including bullous skin diseases, erythema multiforme andcontact dermatitis, psoriasis, allergic diseases such as asthma,allergic rhinitis, atopic dermatitis, food hypersensitivity andurticaria, immunologic diseases of the lung such as eosinophilicpneumonias, idiopathic pulmonary fibrosis and hypersensitivitypneumonitis, transplantation associated diseases including graftrejection and graft-versus-host-disease. Infectious diseases includingviral diseases such as AIDS (HIV infection), herpes, etc., bacterialinfections, fungal infections, protozoal infections, parasiticinfections, and respiratory syncytial virus, human immunodeficiencyvirus, etc.) and allergic disorders, such as anaphylactichypersensitivity, asthma, allergic rhinitis, atopic dermatitis, vernalconjunctivitis, eczema, urticaria and food allergies, etc.

“Treatment” is an intervention performed with the intention ofpreventing the development or altering the pathology of a disorder.Accordingly, “treatment” refers to both therapeutic treatment andprophylactic or preventative measures, wherein the object is to preventor slow down (lessen) the targeted pathological condition or disorder.Those in need of treatment include those already with the disorder aswell as those in which the disorder is to be prevented. In treatment ofa cartilaginous disorder, a therapeutic agent may directly decrease orincrease the magnitude of response of a pathological component of thedisorder, or render the disease more susceptible to treatment by othertherapeutic agents, e.g. antibiotics, antifungals, anti-inflammatoryagents, chemotherapeutics, etc.

The term “effective amount” is the minimum concentration of insulinand/or variant thereof which causes, induces or results in either adetectable improvement or repair in damaged cartilage or a measureableprotection from the continued or induced cartilage destruction in anisolated sample of cartilage matrix (e.g., retention of proteoglycans inmatrix, inhibition of proteoglycan release from matrix, stimulation ofproteoglycan synthesis). Furthermore a “therapeutically effectiveamount” is the minimum concentration (amount) of insulin and/or variantthereof administered to a mammal which would be effective in at leastattenuating a pathological symptom (e.g. causing, inducing or resultingin either a detectable improvement or repair in damaged articularcartilage or causing, inducing or resulting in a measurable protectionfrom the continued or initial cartilage destruction, improvement inrange of motion, reduction in pain, etc.) which occurs as a result ofinjury or a cartilaginous disorder.

“Cartilage agent” may be a growth factor, cytokine, small molecule,antibody, piece of RNA or DNA, virus particle, peptide, or chemicalhaving a beneficial effect upon cartilage, including peptide growthfactors, catabolism antagonists and osteo-, synovial- oranti-inflammatory factors. Alternatively, “cartilage agent” may be apeptide growth factor—such as any of the fibroblast growth factors(e.g., FGF-1, FGF-2, . . . FGF-21, etc.), IGF's (I and II), TGF-βs(1-3), BMPs (1-7), or members of the epidermal growth factor family suchas EGF, HB-EGF, TGF-α—which could enhance the intrinsic reparativeresponse of cartilage, for example by altering proliferation,differentiation, migration, adhesion, or matrix production bychondrocytes. Alternatively, a “cartilage agent” may be a factor whichantagonizes the catabolism of cartilage (e.g., IL-1 receptor antagonist(IL-1ra), NO inhibitors, IL1-β convertase (ICE) inhibitors, factorswhich inhibit activity of IL-6, IL-8, LIF, IFNγ, TNF-α activity,tetracyclines and variants thereof, inhibitors of apoptosis, MMPinhibitors, aggrecanase inhibitors, inhibitors of serine and cysteineproteinases such as cathepsins and urokinase or tissue plasminogenactivator (uPA and tPA). Alternatively still, cartilage agent includesfactors which act indirectly on cartilage by affecting the underlyingbone (i.e., osteofactors, e.g. bisphosphonates or osteoprotegerin) orthe surrounding synovium (i.e., synovial factors) or anti-inflammatoryfactors (e.g., anti-TNF-α, IL1ra, IL-4, IL-10, IL-13, NSAIDs). For areview of cartilage agent examples, please see Martel-Pelletier et al.,Front. Biosci. 4: d694-703 (1999); Hering, T. M., Front. Biosci. 4:d743-761 (1999).

“Standard surgical techniques” are surgical procedures which arecommonly employed for therapeutic manipulations of cartilage, including:cartilage shaving, abrasion chondroplasty, laser repair, debridement,chondroplasty, microfracture with or without subchondral bonepenetration, mosaicplasty, cartilage cell allografts, stem cellautografts, costal cartilage grafts, chemical stimulation, electricalstimulation, perichondral autografts, periosteal autografts, cartilagescaffolds, shell (osteoarticular) autografts or allografts, orosteotomy. These techniques are reviewed and described in better detailin Frenkel et al., Front. Bioscience 4: d671-685 (1999).

An “IGFBP” or an “IGF binding protein” refers to a protein orpolypeptide normally associated with or bound or complexed to IGF-1 orIGF-2, whether or not it is circulatory (ie., in serum or tissue). Suchbinding proteins do not include receptors. This definition includesIGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5, IGFBP-6, Mac 25 (IGFBP-7),and prostacyclin-stimulating factor (PSF) or endothelial cell-specificmolecule (ESM-1), as well as other proteins with high homology toIGFBPs. Mac 25 is described, for example, in Swisshelm et al., Proc.Natl. Acad. Sci. USA, 92: 4472-4476 (1995) and Oh et al., J. Biol.Chem., 271: 30322-30325 (1996). PSF is described in Yamauchi et al.,Biochem. J., 303: 591-598 (1994). ESM-1 is described in Lassalle et al.,J. Biol. Chem., 271: 20458-20464 (1996). For other identified IGFBPs,see, e.g., EP 375,438 published 27 Jun. 1990; EP 369,943 published 23May 1990; WO 89/09268 published 5 Oct. 1989; Wood et al., Molec.Endocrin., 2: 1176-1185 (1988); Brinkman et al., The EMBO J., 7:2417-2423 (1988); Lee et al., Mol. Endocrinol., 2: 404-411 (1988);Brewer et al., BBRC, 152: 1289-1297 (1988); EP 294,021 published 7 Dec.1988; Baxter et al., BBRC, 147: 408-415 (1987); Leung et al., Nature,330: 537-543 (1987); Martin et al., J. Biol. Chem., 261: 8754-8760(1986); Baxter et al., Comp. Biochem. Physiol., 91B: 229-235 (1988); WO89/08667 published 21 Sep. 1989; WO 89/09792 published 19 Oct. 1989; andBinkert et al., EMBO J., 8: 2497-2502 (1989).

“Chronic” administration refers to administration of the factor(s) in acontinuous mode as opposed to an acute mode, so as to maintain theinitial therapeutic effect (activity) for an extended period of time.“Intermittent” administration is treatment that is done notconsecutively without interruption, but rather is cyclic in nature.

The “pathology” of a cartilaginous disorder includes any physiologicalphenomena that compromise the well-being of the patient. This includes,without limitation, cartilage destruction, diminished cartilage repair,abnormal or uncontrollable cell growth or differentiation, antibodyproduction, auto-antibody production, complement production andactivation, interference with the normal functioning of neighboringcells, production of cytokines or other secretory products at abnormallevels, suppression or aggravation of any inflammatory or immunologicalresponse, infiltration of inflammatory cells (neutrophilic,eosinophilic, monocytic, lymphocytic) into tissue spaces, induction ofpain, or any tissue effect which results in impairment of joint functionor mobility etc.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, horses, cats, cattle, pigs, hamsters, etc.Preferably, the mammal is human.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers which are nontoxic to the cell or mammalbeing exposed thereto at the dosages and concentrations employed. Oftenthe physiologically acceptable carrier is an aqueous pH bufferedsolution. Examples of physiologically acceptable carriers includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN®, polyethylene glycol (PEG), and PLURONICS®, hyaluronicacid (HA).

The term “insulin” and/or “insulin variant” when used herein encompassboth (1) native sequence insulin and (2) insulin variants (which arefurther defined herein). The insulin molecule may be isolated from avariety of sources, such as from human tissue types or from anothersource, or prepared by recombinant and/or synthetic methods.

A “native sequence insulin” comprises a polypeptide having the sameamino acid sequence as insulin derived from nature. Such native sequenceinsulin polypeptide can be isolated from nature or can be produced byrecombinant and/or synthetic means. The term “native sequence insulin”specifically encompasses naturally-occurring truncated or secreted forms(e.g., an extracellular domain sequence), naturally-occurring truncatedforms (e.g., alternatively spliced forms) and naturally-occurringallelic variants of insulin. In one embodiment of the invention, thenative sequence human insulin is a mature or full-length native sequenceinsulin comprising an alpha (α) or A chain of amino acids 1 to 21 of SEQID NO: 1 and a beta or B (β) chain of amino acids 1 to 30 of SEQ IDNO:2.

“Insulin variant polypeptide” means an active insulin polypeptide asdefined below having at least about 80% amino acid sequence identitywith the amino acid sequence of: (a) residues 1 to 21 of the A-chain ofthe human insulin polypeptide shown in FIG. 18A (SEQ ID NO:1) incombination with residues 1 to 30 of the B-chain of the human insulinpolypeptide shown in FIG. 18B (SEQ ID NO:2), or (b) another specificallyderived fragment of the amino acid sequences shown in FIGS. 18A and 18B(SEQ ID NO:1-2), or as described herein. In addition, the secondarystructure depicted in FIGS. 18A and 18B, namely the disulfide bondsbetween cysteine residues A6-A11, A7-B7 and A20-B19 are also believednecessary for activity, thus the scope of variants under thisdefinitions should also drawn so as to preserve this secondary structureas much as possible.

Insulin variant polypeptides include, for instance, polypeptides whereinone or more amino acid residues are added, or deleted, at the N- and/orC-terminus, as well as within one or more internal domains, of thesequences shown in FIGS. 18A & 18B (SEQ ID NO:1-2). Ordinarily, aninsulin variant polypeptide will have at least about 80% amino acidsequence identity, alternatively at least about 81% amino acids sequenceidentity, alternatively at least about 82% amino acid sequence identity,alternatively at least about 83% amino acid sequence identity,alternatively at least about 84% amino acid sequence identity,alternatively at least about 85% amino acid sequence identity,alternatively at least about 86% amino acid sequence identity,alternatively at least about 87% amino acid sequence identity,alternatively at least about 88% amino acid sequence identity,alternatively at least about 89% amino acid sequence identity,alternatively at least about 90% amino acid sequence identity,alternatively at least about 91% amino acid sequence identity,alternatively at least about 92% amino acid sequence identity,alternatively at least about 93% amino acid sequence identity,alternatively at least about 94% amino acid sequence identity,alternatively at least about 95% amino acid sequence identity,alternatively at least about 96% amino acid sequence identity,alternatively at least about 97% amino acid sequence identity,alternatively at least about 98% amino acid sequence identity,alternatively at least about 99% amino acid sequence identity with: (a)residues 1 to 21 of the A-chain of the human insulin polypeptide shownin FIG. 18A (SEQ ID NO:1) in combination with residues 1 to 30 of theB-chain of the human insulin polypeptide shown in FIG. 18B (SEQ IDNO:2), or (b) another specifically derived fragment of the amino acidsequences shown in FIGS. 18A and 18B (SEQ ID NO:1-2), or as describedherein.

Insulin variant polypeptides have an A chain length of at least about 15residues, alternatively at least about 16 residues, alternatively atleast about 17 residues, alternatively at least about 18 residues,alternatively at least about 19 residues, alternatively at least about20 residues, alternatively at least about 21 residues, alternatively atleast about 22 residues, alternatively at least about 23 residues,alternatively at least about 24 residues, alternatively at least about25 residues, alternatively at least about 26 residues, alternatively atleast about 27 residues, alternatively at least about 28 residues,alternatively at least about 29 residues, alternatively at least about30 residues, alternatively at least about 35 residues, or more. Insulinvariant polypeptides have an B chain length of at least about 25residues, alternatively at least about 26 residues, alternatively atleast about 27 residues, alternatively at least about 28 residues,alternatively at least about 29 residues, alternatively at least about29 residues, alternatively at least about 30 residues, alternatively atleast about 31 residues, alternatively at least about 32 residues,alternatively at least about 33 residues, alternatively at least about34 residues, alternatively at least about 35 residues, alternatively atleast about 36 residues, alternatively at least about 37 residues,alternatively at least about 38 residues, alternatively at least about39 residues, alternatively at least about 40 residues, alternatively atleast about 41 residues, alternatively at least about 42 residues,alternatively at least about 42 residues, alternatively at least about43 residues, alternatively at least about 44 residues, alternatively atleast about 45 residues, alternatively at least about 50 residues.

The term “proinsulin” and/or “proinsulin variant” when used hereinencompass both native sequence and proinsulin variants (which arefurther defined herein). The proinsulin molecule may be isolated from avariety of sources, such as from human tissue types or from anothersource, or prepared by recombinant and/or synthetic means.

A “native sequence proinsulin” comprises a polypeptide having the sameamino acid sequence as a proinsulin derived from nature. Such nativesequence insulin polypeptide can be isolated from nature or can beproduced by recombinant and/or synthetic means. The term “nativesequence proinsulin” specifically encompasses naturally-occurringtruncated or secreted forms (e.g., an extracellular domain sequence),naturally-occurring truncated forms (e.g., alternatively spliced forms)and naturally-occurring allelic variants of insulin. In one embodimentof the invention, the native sequence human proinsulin is a mature orfull-length native sequence insulin comprising residues 1 to 84 of FIG.18C (SEQ ID NO:3). SEQ ID NO:3 contains three distinct segments: (1) aB-chain of residues 1 to 30 (SEQ ID NO:2); (2) a C-chain (connectingpeptide) of residues 31-63 (SEQ ID NO:4) and an A chain of amino acids64 to 84 (counting from the N-terminal side)(SEQ ID NO:1 ).

“Proinsulin variant” means an insulin precursor polypeptide capable ofbeing processed into a mature insulin which is active, as defined below,comprising at least two distinct regions of contiguous residues; whereinone distinct region has at least about 80% amino acid sequence identitywith the amino acid residues 1 to 21 of the A-chain of the human insulinpolypeptide shown in FIG. 18A (SEQ ID NO:1), and the other distinctregion has at least about 80% amino acid sequence identity with theamino in combination with the amino acid residues 1 to 30 of the B-chainof the human insulin polypeptide shown in FIG. 18B (SEQ ID NO:2), or (b)another specifically derived fragment of the amino acid sequences shownin FIGS. 18A and 18B (SEQ ID NO:1-2), or as described herein.Alternatively, a proinsulin variant will have at least 80% amino acidsequence identity, alternatively at least about with residues 1 to 110of FIG. 18C (SEQ ID NO:3), or another specifically derived fragment ofthe amino acid sequences shown in FIG. 18C (SEQ ID NO:3). Additionalregions may include a lengthened or abbreviated c-peptide and/oradditional sequence 5′- or 3′- to the N-terminal and C-terminalresidues, respectively, which provide for signaling and/or facilitateproper refolding into the mature active form.

In addition, the secondary structure depicted in FIGS. 18A and 18B,namely the disulfide bonds between cysteine residues A6-A11, A7-B7 andA20-B19 are also believed necessary for activity, thus the scope ofvariants under this definition should also drawn so as to preserve thissecondary structure of the mature molecule as much as possible

The length of proinsulin variants is the function of the length of thethree main component parts, namely the A, B and C peptides. The lengthof the C-peptide can vary widely, from as few as no residues (i.e.,deleted entirely) or as many as 50 peptide residues. The length of the Aand B chain components of a proinsulin variant vary similarly asdescribed previously for the full length molecule. Moreover, additionalN-terminal sequence may be added for signaling purposes (e.g., to directthe translation product to the secretory pathway of the host cell) or toenhance or control expression (e.g., promoters, operons, etc.). Thelength of such N-terminal residues may comprise from 1 to 100 residues,including any integer contained within the range (i.e., 1, 2, 3 . . .97, 98, 99, etc.).

“Percent (%) amino acid sequence identity” with respect to thepolypeptide sequences identified herein is defined as the percentage ofamino acid residues in a candidate sequence that are identical with theamino acid residues in a sequence of the insulin polypeptide, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. Alignmentfor purposes of determining percent amino acid sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled inthe art can determine appropriate parameters for measuring alignment,including any algorithms needed to achieve maximal alignment over thefull-length of the sequences being compared. For purposes herein,however, % amino acid sequence identity values are obtained as describedbelow by using the sequence comparison computer program ALIGN-2, whereinthe complete source code for the ALIGN-2 program is provided in Table 1.The ALIGN-2 sequence comparison computer program was authored byGenentech, Inc. and the source code shown in Table 1 has been filed withuser documentation in the U.S. Copyright Office, Washington D.C., 20559,where it is registered under U.S. Copyright Registration No. TXU510087.The ALIGN-2 program is publicly available through Genentech, Inc., SouthSan Francisco, Calif. or may be compiled from the source code providedin Table 1. The ALIGN-2 program should be compiled for use on a UNIXoperating system, preferably digital UNIX V4.0D. All sequence comparisonparameters are set by the ALIGN-2 program and do not vary.

For purposes herein, the % amino acid sequence identity of a given aminoacid sequence A to, with, or against a given amino acid sequence B(which can alternatively be phrased as a given amino acid sequence Athat has or comprises a certain % amino acid sequence identity to, with,or against a given amino acid sequence B) is calculated as follows:100 times the fraction X/Ywhere X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. As examples of % amino acid sequence identitycalculations, Table 2 and Table 3 demonstrate how to calculate the %amino acid sequence identity of the amino acid sequence designated“Comparison Protein” to the amino acid sequence designated “PRO”.

Unless specifically stated otherwise, all % amino acid sequence identityvalues used herein are obtained as described above using the ALIGN-2sequence comparison computer program. However, % amino acid sequenceidentity may also be determined using the sequence comparison programNCBI-BLAST2 (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)).The NCBI-BLAST2 sequence comparison program may be downloaded from orotherwise obtained from the National Institutes of Health, Bethesda,Md., USA 20892. NCBI-BLAST2 uses several search parameters, wherein allof those search parameters are set to default values including, forexample, unmask=yes, strand=all, expected occurrences=10, minimum lowcomplexity length=15/5, multi-pass e-value=0.01, constant formulti-pass=25, dropoff for final gapped alignment=25 and scoringmatrix=BLOSUM62.

In situations where NCBI-BLAST2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:100 times the fraction X/Ywhere X is the number of amino acid residues scored as identical matchesby the sequence alignment program NCBI-BLAST2 in that program'salignment of A and B, and where Y is the total number of amino acidresidues in B. It will be appreciated that where the length of aminoacid sequence A is not equal to the length of amino acid sequence B, the% amino acid sequence identity of A to B will not equal the % amino acidsequence identity of B to A.

Also included within the term “insulin variants” are polypeptides whichin the context of the amino acid sequence identity comparisons performedas described above, include amino acid residues in the sequencescompared that are not only identical, but also those that have similarproperties. These polypeptides are termed “positives”. Amino acidresidues that score a positive value to an amino acid residue ofinterest are those that are either identical to the amino acid residueof interest or are a preferred substitution (as defined in Table 6below) of the amino acid residue of interest. For purposes herein, the %value of positives of a given amino acid sequence A to, with, or againsta given amino acid sequence B (which can alternatively be phrased as agiven amino acid sequence A that has or comprises a certain % positivesto, with, or against a given amino acid sequence B) is calculated asfollows:100 times the fraction X/Ywhere X is the number of amino acid residues scoring a positive value asdefined above by the sequence alignment program ALIGN-2 in thatprogram's alignment of A and B, and where Y is the total number of aminoacid residues in B. It will be appreciated that where the length ofamino acid sequence A is not equal to the length of amino acid sequenceB, the % positives of A to B will not equal the % positives of B to A.

“Insulin variant polynucleotide” or “Insulin variant nucleic acidsequence” means a nucleic acid molecule which encodes an active insulinpolypeptide as defined below and which has at least about 80% nucleicacid sequence identity with a nucleic acid sequence which encodes: (a)amino acid residues 1-21 of the A-chain of the human insulin polypeptideshown in FIG. 18A (SEQ ID NO:1) in combination with residues 1 to 30 ofthe B-chain of the human insulin polypeptide shown (with substitution ofthe human residue B30Thr) in FIG. 18B (SEQ ID NO:2), or (b) a nucleicacid sequence which encodes another specifically derived fragment of theamino acid sequence shown in FIGS. 18A and B (SEQ ID NO:1-2).Ordinarily, an insulin variant polynucleotide will have at least about80% nucleic acid sequence identity, more preferably at least about 81%nucleic acid sequence identity, more preferably at least about 82%nucleic acid sequence identity, more preferably at least about 83%nucleic acid sequence identity, more preferably at least about 84%nucleic acid sequence identity, more preferably at least about 85%nucleic acid sequence identity, more preferably at least about 86%nucleic acid sequence identity, more preferably at least about 87%nucleic acid sequence identity, more preferably at least about 88%nucleic acid sequence identity, more preferably at least about 89%nucleic acid sequence identity, more preferably at least about 90%nucleic acid sequence identity, more preferably at least about 91%nucleic acid sequence identity, more preferably at least about 92%nucleic acid sequence identity, more preferably at least about 93%nucleic acid sequence identity, more preferably at least about 94%nucleic acid sequence identity, more preferably at least about 95%nucleic acid sequence identity, more preferably at least about 96%nucleic acid sequence identity, more preferably at least about 97%nucleic acid sequence identity, more preferably at least about 98%nucleic acid sequence identity and yet more preferably at least about99% nucleic acid sequence identity with a nucleic acid sequence encodingamino acid residues: (a) amino acid residues 1-21 of the A-chain of thehuman insulin polypeptide shown in FIG. 18A (SEQ ID NO:1) in combinationwith residues 1 to 30 of the B-chain of the human insulin polypeptideshown (with substitution of the human residue B30Thr) in FIG. 18B (SEQID NO:2), or (b) a nucleic acid sequence which encodes anotherspecifically derived fragment of the amino acid sequence shown in FIGS.18 and B (SEQ ID NO:1-2).

Ordinarily, insulin variant polynucleotides contain nucleic acidencoding an A chain of insulin of at least about 45 nucleotides,alternatively at least about 48 nucleotides, alternatively at leastabout 51 nucleotides, alternatively at least about 54 nucleotides,alternatively at least about 57 nucleotides, alternatively at leastabout 60 nucleotides, alternatively at least about 63 nucleotides,alternatively at least about 66 nucleotides, alternatively at leastabout 69 nucleotides, alternatively at least about 72 nucleotides,alternatively at least about 75 nucleotides, alternatively at leastabout 78 nucleotides, alternatively at least about 81 nucleotides,alternatively at least about 84 nucleotides, alternatively at leastabout 87 nucleotides, alternatively at least about 90 nucleotides,alternatively at least about 105 nucleotides. Insulin variantpolynucleotides contain nucleic acid encoding a B chain of insulin of atleast about 75 nucleotides, alternatively at least about 78 nucleotides,alternatively at least about 81 nucleotides, alternatively at leastabout 84 nucleotides, alternatively at least about 87 nucleotides,alternatively at least about 90 nucleotides, alternatively at leastabout 93 nucleotides, alternatively at least about 96 nucleotides,alternatively at least about 99 nucleotides, alternatively at leastabout 102 nucleotides, alternatively at least about 105 nucleotides,alternatively at least about 108 nucleotides, alternatively at leastabout 111 nucleotides, alternatively at least about 114 nucleotides,alternatively at least about 117 nucleotides, alternatively at leastabout 120 nucleotides, alternatively at least about 123 nucleotides,alternatively at least about 126 nucleotides, alternatively at leastabout 129 nucleotides, alternatively at least about 132 nucleotides,alternatively at least about 135 nucleotides, alternatively at leastabout 150 nucleotides.

“Percent (%) nucleic acid sequence identity” with respect to the insulinpolypeptide-encoding nucleic acid sequences identified herein is definedas the percentage of nucleotides in a candidate sequence that areidentical with the nucleotides in an invention polypeptide-encodingsequence of interest, after aligning the sequences and introducing gaps,if necessary, to achieve the maximum percent sequence identity.Alignment for purposes of determining percent nucleic acid sequenceidentity can be achieved in various ways that are within the skill inthe art, for instance, using publicly available computer software suchas BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Thoseskilled in the art can determine the appropriate parameters formeasuring alignment, including any algorithms needed to achieve maximalalignment over the full-length of the sequences being compared. Forpurposes herein, however, % nucleic acid sequence identity values areobtained as described below by using the sequence comparison computerprogram ALIGN-2, wherein the complete source code for the ALIGN-2program is provided in Table 1. The ALIGN-2 sequence comparison computerprogram was authored by Genentech, Inc. and the source code shown inTable 1 has been filed with user documentation in the U.S. CopyrightOffice, Washington D.C., 20559, where it is registered under U.S.Copyright Registration No. TXU510087. The ALIGN-2 program is publiclyavailable through Genentech, Inc., South San Francisco, Calif. or may becompiled from the source code provided in Table 1. The ALIGN-2 programshould be compiled for use on a UNIX operating system, preferablydigital UNIX V4.0D. All sequence comparison parameters are set by theALIGN-2 program and do not vary.

For purposes herein, the % nucleic acid sequence identity of a givennucleic acid sequence C to, with, or against a given nucleic acidsequence D (which can alternatively be phrased as a given nucleic acidsequence C that has or comprises a certain % nucleic acid sequenceidentity to, with, or against a given nucleic acid sequence D) iscalculated as follows:100 times the fraction W/Zwhere W is the number of nucleotides scored as identical matches by thesequence alignment program ALIGN-2 in that program's alignment of C andD, and where Z is the total number of nucleotides in D. It will beappreciated that where the length of nucleic acid sequence C is notequal to the length of nucleic acid sequence D, the % nucleic acidsequence identity of C to D will not equal the % nucleic acid sequenceidentity of D to C. As examples of % nucleic acid sequence identitycalculations, Tables 4 and 5 demonstrate how to calculate the % nucleicacid sequence identity of the nucleic acid sequence designated“Comparison DNA” to the nucleic acid sequence designated “PRO-DNA”.

Unless specifically stated otherwise, all % nucleic acid sequenceidentity values used herein are obtained as described above using theALIGN-2 sequence comparison computer program. However, % nucleic acidsequence identity may also be determined using the sequence comparisonprogram NCBI-BLAST2 (Altschul et al., Nucleic Acids Res. 25:3389-3402(1997)). The NCBI-BLAST2 sequence comparison program may be downloadedfrom or otherwise obtained from the National Institutes of Health,Bethesda, Md. USA 20892. NCBI-BLAST2 uses several search parameters,wherein all of those search parameters are set to default valuesincluding, for example, unmask=yes, strand=all, expected occurrences=10,minimum low complexity length=15/5, multi-pass e-value=0.01, constantfor multi-pass=25, dropoff for final gapped alignment=25 and scoringmatrix=BLOSUM62.

In situations where NCBI-BLAST2 is employed for sequence comparisons,the % nucleic acid sequence identity of a given nucleic acid sequence Cto, with, or against a given nucleic acid sequence D (which canalternatively be phrased as a given nucleic acid sequence C that has orcomprises a certain % nucleic acid sequence identity to, with, oragainst a given nucleic acid sequence D) is calculated as follows:100 times the fraction W/Zwhere W is the number of nucleotides scored as identical matches by thesequence alignment program NCBI-BLAST2 in that program's alignment of Cand D, and where Z is the total number of nucleotides in D. It will beappreciated that where the length of nucleic acid sequence C is notequal to the length of nucleic acid sequence D, the % nucleic acidsequence identity of C to D will not equal the % nucleic acid sequenceidentity of D to C.

In other embodiments, invention variant polynucleotides are nucleic acidmolecules that encode an active polypeptide of the invention and whichare capable of hybridizing, preferably under stringent hybridization andwash conditions, to nucleotide sequences encoding the full-lengthinvention polypeptide. Invention variant polypeptides include those thatare encoded by an invention variant polynucleotide.

The term “positives”, in the context of the amino acid sequence identitycomparisons performed as described above, includes amino acid residuesin the sequences compared that are not only identical, but also thosethat have similar properties. Amino acid residues that score a positivevalue to an amino acid residue of interest are those that are eitheridentical to the amino acid residues of interest or are a preferredsubstitution (as defined in Table 6 below) of the amino acid residue ofinterest.

For purposes herein, the % value of positives of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % positives to, with, or against a given amino acidsequence B) is calculated as follows:100 times the fraction X/Ywhere X is the number of amino acid residues scoring a positive value asdefined above by the sequence alignment program ALIGN-2 in thatprogram's alignment of A and B, and where Y is the total number of aminoacids residues in B. It will be appreciated that where the length ofamino acid sequence A is not equal to the length of amino acid sequenceB, the % positives of A to B will not equal the % positives of B to A.

An “isolated” nucleic acid molecule encoding a insulin or insulinvariant polypeptide is a nucleic acid molecule that is identified andseparated from at least one contaminant nucleic acid molecule with whichit is ordinarily associated in the natural source of the insulin orinsulin variant-encoding nucleic acid. Preferably, the isolated nucleicacid is free of association with all components with which it isnaturally associated. An isolated insulin or insulin variant-encodingnucleic acid molecule is other than in the form or setting in which itis found in nature. Such isolated nucleic acid molecules therefore aredistinguished from the insulin or insulin variant-encoding nucleic acidmolecule as it exists in natural cells. However, an isolated nucleicacid molecule encoding an insulin or insulin variant polypeptideincludes insulin or insulin variant-encoding nucleic acid molecules,respectively, contained in cells that ordinarily express such moleculeswhere, for example, the nucleic acid molecule is in a chromosomallocation different from that of natural cells.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridizable sequence, the higher the relativetemperature which can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

“Stringent conditions” or “high stringency conditions”, as definedherein, may be identified by those that: (1) employ low ionic strengthand high temperature for washing, for example 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3)employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mMsodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt'ssolution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10%dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodiumchloride/sodium citrate) and 50% formamide at 55° C., followed by ahigh-stringency wash consisting of 0.1×SSC containing EDTA at 55° C.

“Moderately stringent conditions” may be identified as described bySambrook et al., Molecular Cloning: A Laboratory Manual, New York: ColdSpring Harbor Press, 1989, and include the use of washing solution andhybridization conditions (e.g., temperature, ionic strength and % SDS)less stringent that those described above. An example of moderatelystringent conditions is overnight incubation at 37° C. in a solutioncomprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextransulfate, and 20 μg/mL denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 37-50° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

The term “epitope tagged” when used herein refers to a chimericpolypeptide comprising a polypeptide of the invention fused to a “tagpolypeptide”. The tag polypeptide has enough residues to provide anepitope against which an antibody can be made, yet is short enough suchthat it does not interfere with the activity of the polypeptide to whichit is fused. The tag polypeptide preferably also is fairly unique sothat the antibody does not substantially cross-react with otherepitopes. Suitable tag polypeptides generally have at least six aminoacid residues and usually between about 8 and 50 amino acid residues(preferably, between about 10 and 20 amino acid residues).

“Active” or “activity” in the context of variants of the polypeptide ofthe invention refers to form(s) of proteins of the invention whichretain the biologic and/or immunologic activities of a native ornaturally-occurring insulin polypeptide, wherein “biological” activityrefers to a biological function (either inhibitory or stimulatory)caused by a native or naturally-occurring insulin other than the abilityto serve as an antigen in the production of an antibody against anantigenic epitope possessed by a native or naturally-occurringpolypeptide of the invention. Similarly, an “immunological” activityrefers to the ability to serve as an antigen in the production of anantibody against an antigenic epitope possessed by a native ornaturally-occurring polypeptide of the invention.

“Biological activity” in the context of an antibody or another moleculethat can be identified by the screening assays disclosed herein (e.g. anorganic or inorganic small molecule, peptide, etc.) is used to refer tothe ability of such molecules to promote the regeneration of and/orprevent the destruction of cartilage. Optionally, the cartilage isarticular cartilage and the regeneration and/or destruction of thecartilage is associated with an injury or a cartilaginous disorder. Forexample, biological activity may be quantified by the inhibition ofproteoglycan (PG) release from articular cartilage, the increase in PGsynthesis in articular cartilage, the inhibition of the production ofNO, etc.

A “small molecule” is defined herein to have a molecular weight belowabout 600 daltons, and is generally an organic compound.

The term “isolated” when it refers to the various polypeptides of theinvention means a polypeptide which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the polypeptide ofthe invention will be purified (1) to greater than 95% by weight of thecompound as determined by the Lowry method, and most preferably morethan 99% by weight, (2) to a degree sufficient to obtain at least 15residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (3) to homogeneity by SDS-PAGE underreducing or nonreducing conditions using Coomassie blue or, preferably,silver stain. Isolated compound, e.g. antibody or polypeptide, includesthe compound in situ within recombinant cells since at least onecomponent of the compound's natural environment will not be present.Ordinarily, however, isolated compound will be prepared by at least onepurification step.

The word “label” when used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly to the compound,e.g. antibody or polypeptide, so as to generate a “labelled” compound.The label may be detectable by itself (e.g. radioisotope labels orfluorescent labels) or, in the case of an enzymatic label, may catalyzechemical alteration of a substrate compound or composition which isdetectable. The word “instruction” by contrast, as used herein refers tolanguage affixed to the packaging of containers indicating a use inconformity with the claimed methods.

By “solid phase” is meant a non-aqueous matrix to which the compound ofthe present invention can adhere. Examples of solid phases encompassedherein include those formed partially or entirely of glass (e.g.,controlled pore glass), polysaccharides (e.g., agarose),polyacrylamides, polystyrene, polyvinyl alcohol and silicones. Incertain embodiments, depending on the context, the solid phase cancomprise the well of an assay plate; in others it is a purificationcolumn (e.g., an affinity chromatography column). This term alsoincludes a discontinuous solid phase of discrete particles, such asthose described in U.S. Pat. No. 4,275,149.

A “liposome” is a small vesicle composed of various types of lipids,phospholipids and/or surfactant which is useful for delivery of a drug(such as the insulin and insulin variants disclosed herein) to a mammal.The components of the liposome are commonly arranged in a bilayerformation, similar to the lipid arrangement of biological membranes.

As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the binding specificity of a heterologousprotein (an “adhesin”) with the effector functions of immunoglobulinconstant domains. Structurally, the immunoadhesins comprise a fusion ofan amino acid sequence with the desired binding specificity which isother than the antigen recognition and binding site of an antibody(i.e., is “heterologous”), and an immunoglobulin constant domainsequence. The adhesin part of an immunoadhesin molecule typically is acontiguous amino acid sequence comprising at least the binding site of areceptor or a ligand. The immunoglobulin constant domain sequence in theimmunoadhesin may be obtained from any immunoglobulin, such as IgG-1,IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE,IgD or IgM.

The term “extended-release” or “sustained-release” formulations in thebroadest possible sense means a formulation of active insulin or insulinvariant polypeptide resulting in the release or activation of the activepolypeptide for a sustained or extended period of time—or at least for aperiod of time which is longer than if the polypeptide was madeavailable in vivo in the native or unformulated state. Optionally, theextended-release formulation occurs at a constant rate and/or results insustained and/or continuous concentration of the active polypeptide.Suitable extended release formulations may comprise microencapsulation,semi-permeable matrices of solid hydrophobic polymers, biodegradablepolymers, biodegradable hydrogels, suspensions or emulsions (e.g.,oil-in-water or water-in-oil). Optionally, the extended-releaseformulation comprises poly-lactic-co-glycolic acid (PLGA) and can beprepared as described in Lewis, “Controlled Release of Bioactive Agentsform Lactide/Glycolide polymer,” in Biodegradable Polymers as DrugDelivery Systems, M. Chasin & R. Langeer, Ed. (Marcel Dekker, New York),pp. 1-41. Optionally, the extended-release formulation is stable and theactivity of the insulin or insulin variant does not appreciably diminishwith storage over time. More specifically, such stability can beenhanced through the presence of a stabilizing agent such as awater-soluble polyvalent metal salt.

Table 1 below provides the complete source code for the ALIGN-2 sequencecomparison computer program. This source code may be routinely compiledfor use on a UNIX operating system to provide the ALIGN-2 sequencecomparison computer program.

Tables 2-5 below shows hypothetical exemplifications for using the belowdescribed method to determine % amino acid sequence identity (Tables2-3) and % nucleic acid sequence identity (Tables 4-5) using the ALIGN-2sequence comparison computer program, wherein “PRO” represents the aminoacid sequence of a hypothetical polypeptide of the invention ofinterest, “Comparison Protein” represents the amino acid sequence of apolypeptide against which the “PRO” polypeptide of interest is beingcompared, “PRO-DNA” represents a hypothetical “PRO”-encoding nucleicacid sequence of interest, “Comparison DNA” represents the nucleotidesequence of a nucleic acid molecule against which the “PRO-DNA” nucleicacid molecule of interest is being compared, “X, “Y” and “Z” eachrepresent different hypothetical amino acid residues and “N”, “L” and“V” each represent different hypothetical nucleotides.

TABLE 2 PRO XXXXXXXXXXXXXXX (Length = 15 amino acids) ComparisonXXXXXYYYYYYY (Length = 12 amino acids) Protein % amino acid sequenceidentity = (the number of identically matching amino acid residuesbetween the two polypeptide sequences as determined by ALIGN-2) dividedby (the total number of amino acid residues of the PRO polypeptide) = 5divided by 15 = 33.3%

TABLE 3 PRO XXXXXXXXXX (Length = 10 amino acids) ComparisonXXXXXYYYYYYZZYZ (Length = 15 amino acids) Protein % amino acid sequenceidentity = (the number of identically matching amino acid residuesbetween the two polypeptide sequences as determined by ALIGN-2) dividedby (the total number of amino acid residues of the PRO polypeptide) = 5divided by 10 = 50%

TABLE 4 PRO-DNA NNNNNNNNNNNNNN (Length = 14 nucleotides) ComparisonNNNNNNLLLLLLLLLL (Length = 16 nucleotides) DNA % nucleic acid sequenceidentity = (the number of identically matching nucleotides between thetwo nucleic acid sequences as determined by ALIGN-2) divided by (thetotal number of nucleotides of the PRO-DNA nucleic acid sequence) = 6divided by 14 = 42.9%

TABLE 5 PRO-DNA NNNNNNNNNNNN (Length = 12 nucleotides) ComparisonNNNNLLLVV (Length = 9 nucleotides) DNA % nucleic acid sequence identity= (the number of identically matching nucleotides between the twonucleic acid sequences as determined by ALIGN-2) divided by (the totalnumber of nucleotides of the PRO-DNA nucleic acid sequence) = 4 dividedby 12 = 33.3%II. Modes for Carrying Out the Invention

A. Articular Cartilage Explant Assay

In this assay, the synthetic and prophylactic potential of the testcompound on intact cartilage is described. To this end, proteoglycan(PG) synthesis and breakdown, and nitric oxide release are measured intreated articular cartilage explants. Proteoglycans are the secondlargest component of the organic material in articular cartilage(Kuettner, K. E. et al., Articular Cartilage Biochemistry, Raven Press,New York, USA (1986), p. 456; Muir, H., Biochem. Soc. Tran. 11: 613-622(1983); Hardingham, T. E., Biochem. Soc. Trans. 9: 489-497 (1981). Sinceproteoglycans help determine the physical and chemical properties ofcartilage, the decrease in cartilage PGs which occurs during jointdegeneration leads to loss of compressive stiffness and elasticity, anincrease in hydraulic permeability, increased water content (swelling),and changes in the organization of other extracellular components suchas collagens. Thus, PG loss is an early step in the progression ofcartilaginous disorders, one which further perturbs the biomechanicaland biochemical stability of the joint. PGs in articular cartilage havebeen extensively studied because of their likely role in skeletal growthand disease. Mow, V. C., & Ratcliffe, A. Biomaterials 13: 67-97 (1992).Proteoglycan breakdown, which is increased in diseased joints, ismeasured in the assays described herein by quantitating PGs releasedinto the media by articular cartilage explants using the colorimetricDMMB assay. Farndale and Buttle, Biochem. Biophys. Acta 883: 173-177(1985). Incorporation of ³⁵S-sulfate into proteoglycans is used tomeasure proteoglycan synthesis.

The evidence linking interleukin-1α, IL-1α, and degenerativecartilaginous disorders is substantial. For example, high levels ofIL-1α (Pelletier J P et al., “Cytokines and inflammation in cartilagedegradation” in Osteoarthritic Edition of Rheumatic Disease Clinics ofNorth America, Eds. R W Moskowitz, Philadelphia, W.D. Saunders Company,1993, p. 545-568) and IL-1 receptors (Martel-Pelletier et al., ArthritisRheum. 35: 530-540 (1992) have been found in diseased joints, and IL-1αinduces cartilage matrix breakdown and inhibits synthesis of new matrixmolecules. Baragi et al., J. Clin. Invest. 96: 2454-60 (1995); Baragi etal., Osteoarthritis Cartilage 5: 275-82 (1997); Evans et al., J. Leukoc.Biol. 64: 55-61 (1998); Evans et al., J. Rheumatol. 24: 2061-63 (1997);Kang et al., Biochem. Soc. Trans. 25: 533-37 (1997); Kang et al.,Osteoarthritis Cartilage 5: 139-43 (1997). Because of the association ofIL-1α with disease, the test compound is also assayed in the presence ofIL-1α. The ability of the test compound to not only have positiveeffects on cartilage, but also to counteract the catabolic effects ofIL-1α is strong evidence of the protective effect exhibited by the testcompound. In addition, such and activity suggests that the test compoundcould inhibit the degradation which occurs in arthritic conditions,since catabolic events initiated by IL-1α are also induced by many othercytokines and since antagonism of IL-1α activity has been shown toreduce the progression of osteoarthritis. Arend, W. P. et al., Ann. Rev.Immunol. 16: 27-55 (1998).

The production of nitric oxide (NO) can be induced in cartilage bycatabolic cytokines such as IL-1. Palmer, R M J et al., Biochem.Biophys. Res. Commun. 193: 398-405 (1993). NO has also been implicatedin the joint destruction which occurs in arthritic conditions. Ashok etal., Curr. Opin. Rheum. 10: 263-268 (1998). Unlike normal (undiseased oruninjured) cartilage, osteoarthritic cartilage produced significantamounts of nitric oxide ex vivo, even in the absence of added stimulisuch as interleukin-1 or lipopolysaccharide (LPS). In vivo animal modelssuggest that inhibition of nitric oxide production reduces progressionof arthritis. Pelletier, J P et al., Arthritis Rheum. 7: 1275-86 (1998);van de Loo et al., Arthritis Rheum. 41: 634-46 (1998); Stichtenoth, D.O. and Frolich J. C., Br. J. Rheumatol. 37: 246-57 (1998). In vitro,nitric oxide exerts detrimental effects on chondrocyte function,including inhibition of collagen and proteoglycan synthesis, inhibitionof adhesion to the extracellular matrix, and enhancement of cell death(apoptosis). Higher concentrations of nitrite are found in synovialfluid from osteoarthritic patients than in fluid from rheumatoidarthritic patients. Renoux et al., Osteoarthritis Cartilage 4: 175-179(1996). Furthermore, animal models suggest that inhibition of nitricoxide production reduces progression of arthritis. Pelletier, J. P. etal., Arthritis Rheum. 7: 1275-86 (1998); van de Loo et al., ArthritisRheum. 41: 634-46 (1998); Stichtenoth, D. O. & Frolich, J. C., Br. J.Rheumatol. 37: 246-57 (1998). Since NO also has effects on other cells,the presence of NO within the articular joint could increasevasodilation and permeability, potentiate cytokine release byleukocytes, and stimulate angiogenic activity. Since NO likely play arole in both the erosive and the inflammatory components of jointdiseases, a factor which decreases nitric oxide production would likelybe beneficial for the treatment of cartilaginous disorders.

The assay to measure nitric oxide production described herein is basedon the principle that 2,3-diaminonapthalene (DAN) reacts with nitriteunder acidic conditions to form 1-(H)-naphthotriazole, a fluorescentproduct. As NO is quickly metabolized into nitrite (NO₂ ⁻¹) and nitrate(NO₃ ⁻¹), detection of nitrite is one means of detecting (albeitundercounting) the actual NO produced by cartilage.

The procedures employed are described in greater detail in the examples.

B. Maintenance of Chondrocytes in Serum-free Culture

In this procedure, the ability of the test compound to enhance, promoteor maintain the viability of chondrocytes in cultures in the absence ofserum or other growth factors is examined. Articular chondrocytes arefirst prepared by removal of the extracellular matrix and cultured in amonolayer, which is believed to approximate the latter stages ofcartilage disorders when the matrix has been depleted.

The assay is a colorimetric assay that measures the metabolic activityof the cultured cells based on the ability of viable cells to cleave theyellow tetrazolium salt MTT to form purple formazan crystals. Thiscellular reduction reaction involves the pyridine nucleotide cofactorsNADH and NADPH. Berridge, M. V. & Tan, A. S., Arch. Biochem. Biophys.303: 474 (1993). The solubilized product is spectrophotometricallyquantitated on an ELISA reader. The procedure is described in greaterdetail in the examples.

C. Mouse Patellae Assay

The experiment examines the effects of the test compounds onproteoglycan synthesis in patellae (kneecaps) of mice. This assay usesintact cartilage (including the underlying bone) and thus tests factorsunder conditions which approximate the in vivo environment of cartilage.Compounds are either added to patellae in vitro, or are injected intoknee joints in vivo prior to analysis of proteoglycan synthesis inpatellae ex vivo. As has been shown previously, in vivo treated patellaeshow distinct changes in PG synthesis ex vivo (Van den Berg et al.,Rheum. Int. 1: 165-9 (1982); Vershure, P. J. et al., Ann. Rheum. Dis.53: 455-460 (1994); and Van de Loo et al., Arthrit. Rheum. 38: 164-172(1995). In this model, the contralateral joint of each animal can beused as a control. The procedure is better described in the examples.

D. Guinea Pig Model

These experiments measure the effects of the test compound on both thestimulation of PG synthesis and inhibition of PG release in articularcartilage explants from a strain of guinea pigs, Dunkin Hartley (DH),which spontaneously develops knee osteoarthritis (OA). Most other animalmodels which cause rapidly progressing joint breakdown resemblesecondary OA more than the slowly evolving human primary OA. Incontrast, DH guinea pigs have naturally occurring slowly progressive,non-inflammatory OA-like changes. Because the highly reproduciblepattern of cartilage breakdown in these guinea pigs is similar to thatseen in the human disorder, the DH guinea pig is a well-accepted animalmodel for osteoarthritis. Young et al., “Osteoarthrits”, Spontaneousanimal models of human disease vol. 2, pp. 257-261, Acad. Press, NewYork. (1979); Bendele et al., Arthritis Rheum. 34: 1180-1184; Bendele etal., Arthritis Rheum. 31: 561-565 (1988); Jimenez et al., LaboratoryAnimal Sciences 47 (6): 598-601 (1997); Wei et al., Acta Orthop Scand69: 351-357 (1998)). Initially, these animals develop a mild OA that isdetectable by the presence of minimal histologic changes. However, thedisease progresses, and by 16-18 months of age, moderate to severecartilage degeneration within the joints is observed (FIG. 8).

As a result, the effect of the test compound on the cartilage matrix ofthe DH guinea pigs over the progression of the disease would beindicative of the therapeutic effect of the compound in the treatment ofOA at different stages of joint destruction.

The procedure is described in better detail in the examples.

E. Diabetic Mouse Model

The metabolic changes associated with diabetes mellitus (diabetes)affect many other organ and musculo-skeletal systems of the afflictedorganism. For example, in humans, the incidence of musculoskeletalinjuries and disorders is increased with the onset of diabetes, anddiabetes is considered a risk factor for the development of arthritis.

A syndrome similar to diabetes can be induced in animals byadministration of streptozotocin (STZ). Portha B. et al., Diabete Metab.15: 61-75 (1989). By killing pancreatic cells which produce insulin, STZdecreases the amount of serum insulin in treated animals. STZ-induceddiabetes is associated with atrophy and depressed collagen content ofconnective tissues including skin, bone and cartilage. Craig, R. G. etal., Biochim. Biophys. Acta 1402: 250-260 (1998).

In this procedure, the patellae of treated STZ-treated mice areincubated in the presence of the test compound and the resulting matrixsynthesis is analyzed. The ability of the test compound to increase orrestore the level of PG synthesis to that of untreated controls isindicative of the therapeutic potential. The procedure is betterdescribed in the examples.

F. Extended Release Formulation as Polymeric Microspheres

While intermittent injections are generally well-tolerated by patientsand once/week injections of therapeutics are currently being testedclinically, an ideal drug would be one in which a limited number ofdoses was required. However, when the active compound is unstable or isquickly degraded at physiological conditions, a stabilized, slow-releaseformulation is highly desirable.

These experiments, described in greater detail as Examples 6-8 examinethe effectiveness of a slow-release formulation of the tested compoundas indicated by (1) the size, protein load and physical integrity; (2)the release profile of the test compound from the slow-release matrix;and (3) the biological activity of the test compound after release fromthe slow-release matrix.

These procedures are better described in the examples below.

1. Physical Characteristics

Under some circumstances, it may be first necessary or desirable tostabilize the test compound with other complexing or stabilizing agentsprior to incorporation into the slow-release form. For example, whilethe particular compound of the invention human insulin (HI) would appearto be very effective in treating cartilaginous disorders, it is unstablewhen stored in neutral conditions at low concentrations for extendedperiods of time. J. Brenge and L. Langkjoer, Insulin Formulation andDelivery in Protein Delivery, Eds, L M. Sanders and W. Hendren, PlenumPress, 1997. Moreover, HI is quickly degraded in the body, having ahalf-life of only 5 minutes in the human body. Hadley, ME,Endocrinology, Prentice-Hall, Inc. 1988.

One potential solution to the problem of a short life and instabilitythat has been attempted with HI is formulation with zinc. A sparinglysoluble zinc acetate:HI formulation has been attempted elsewhere, basedon histochemical evidence suggesting that insulin is stored in thepancreas in a complex with zinc. Eli Lilly, Indianapolis, Ind.; J.Brenge and L. Langkjoer, supra.; Hadley, supra. Other evidence indicatesthat HI complexed with zinc is more resistant to aggregation and has aslower onset and longer duration of activity relative to uncomplexedmaterial. J. Brenge & L. Langkjoer, supra.

The slow-release formulation described in this procedure comprisesmicroencapsulation of a spray-freeze dried compound into a polylactic-coglycolic acid (PLGA) matrix using the procedure as described byGombotz et al., U.S. Pat. No. 5,019,400 and Johnson et al., Nature Med.2(7):795-799 (1996).

In this procedure, the amount of test compound in the slow-releasecomposition was determined by chemical analysis, while the physical andbiological integrity of the test material recoverable from thecompositions were determined by size-exclusion (SEC) and reverse phasechromatography (RPC).

2. Release Profiles

In this procedure, the insulin-loaded PLGA microspheres were incubatedin 3 different conditions and the recovered protein was analyzed foractivity at several timepoints. In this slow-release system, testcompound is released from the microsphere by treatment with sodiumhydroxide and/or histidine buffer. In order to better approximatephysiological conditions, the microspheres are also incubated in eithersynovial fluid or with articular cartilage explants.

The release of the test compound from the microsphere is determinable byany method which is typically used for assaying for the presence and/oractivity of the test compound. For example, with HI, a suitabletechnique is the use of an insulin receptor kinase assay (KIRA) in cellsexpressing the insulin receptor (e.g., CHO cells).

3. Biological Activity

In this procedure the biological activity of the test compound releasedfrom microencapsulation is determined. Even though the procedure of thelast section was a measure of biological activity of the test compoundin the generic sense, it is important to confirm that the released testcompound still retains a particular desired biological activity onarticular cartilage (e.g., stimulation of matrix synthesis andinhibition of matrix breakdown).

G. Human Articular Cartilage Explant Assay

In a manner similar to that described above under articular cartilageexplant assay, this procedure measures the anabolic effects of the testmolecule, except that the tissue source is human. The procedure isdescribed in greater detail in Example 9.

III. Compositions and Methods of the Invention

A. Full-Length Insulin and/or Variants Thereof

The present invention provides in part a novel method for using insulinand insulin variant polypeptides to treat cartilaginous disorders,including regenerating and/or preventing the degradation of cartilage.In particular, cDNAs encoding insulin and insulin variant polypeptideshave been identified, isolated and their use in the treatment ofcartilaginous disorders is disclosed herein. Insulin is a well-knownmolecule and recombinant human insulin is readily available fromnumerous commercial suppliers including Lilly, Novo-Nordisk, and Sigma.Alternatively the insulin and insulin variant molecules for use with thepresent invention may be obtained by any known technique from thepolypeptide sequence identified as SEQ ID NO:1 & 2 (FIGS. 18A & 18B).

Native human insulin has two peptide chains, an A chain containing 21amino acids (SEQ ID NO:1) and a B chain containing 30 amino acidresidues (SEQ ID NO:2). The two chains contain 3 disulfide bridges, eachformed of two cysteinyl residues. Two of the bridges are interchainbridges, between residues A7-B7 and A-20-B-19, and the other is anintrachain bridge between residues A-6-A-11 (FIGS. 17A-B).

Insulin may be produced be the separate expression of the A and Bchains, followed by a refolding reaction to form the disulfide bonds.Chance et al., Diabetes Care 4: 147-154 (1982). These chains may beexpressed in E. coli as β-galactosidase fusion proteins. Williams etal., Science 215(5): 687-689 (1982). The separate chains (S-sulfonatedforms) can be combined to in the presence of mercaptan to obtain theactive molecule. Goeddel et al., Proc. Natl. Acad. Sci. U.S.A. 76(1):106-110 (1979). Because of the large size of the β-gal fusion protein(i.e. over 1000 amino acids), premature detachment from the ribosomeoften occurs, resulting in incomplete insulin translations. Burnett,Experimental Manipulation of Gene Expression, Inouye, Ed. AcademicPress, New York, pp 259-277 (1983). An improvement in this procedure isthe use of the tryptophan (Trp) operon in place of the lac operon in theβ-gal expression system. Hall, Invisible Frontiers—The Race toSynthesize a Human Gene, Atlantic Monthly Press, New York, 1987). TheTrp operon is a series of five bacterial genes which sequentiallysynthesize the enzymes responsible for the anabolism of tryptophan. TheTrp operon offers several advantages: (1) the Trp E only has about 190amino acid residues as compared to the β-gal enzyme (1000), thus greatlyreducing the probability of premature chain termination; (2) the Trp Egene increases expression of the fusion protein, resulting in 10-foldgreater expression compared to the lac (i.e., β-gal) system; (3) the Trpoperon is activated when the fermentation media is deficient intryptophan, thus, expression can be turned on when host cell mass is ata maximum by allowing the fermentation media to become depleted intryptophan. When the fermentation is complete, the protein is recoveredby disruption of the cell walls and recovery of the inclusion bodies,followed by CNBr cleavage to release to A or B chains and purified byion exchange, size exclusion and reversed-phase high-performance liquidchromatography to obtain the purified recombinant product. Frank andChase, Munch Med. Wschr. 125 (Suppl. 1); 514-520(1983).

Another common method of producing insulin involves the production ofproinsulin. Native sequence proinsulin (SEQ ID NO:3)(FIG. 18C) is asingle chain polypeptide in which the N-terminus of the insulin A-chainis linked through a connecting peptide with the C-terminus of theinsulin B-chain, and the appropriate cysteinyl residues are joined bydisulfide bonds. Human native sequence proinsulin has 86 amino acidresidues, 35 of which make up the connecting peptide, sometimes known asthe C-peptide (SEQ ID NO:4)(FIG. 18D). Yanaihara et al., Diabetes 27(Suppl. 1): 149-160 (1978). The principal importance of the C-peptide isto facilitate the formation of the proper disulfide bridging and/or thetrypsin-like processing at the site of two adjacent basic amino acids.Bell et al., Nature 284: 26-32 (1980); Thim et al., PNAS 83: 6766-6770(1986). This connecting peptide is removeable through enzymaticdigestion. W. Kemmler et al., J. Biol. Chem. 246: 6786 (1971).

Production of proinsulin is described in Kroeff et al., J. Chromatogr.481: 45-61 (1989). Expression may be carried in E. coli by linking amethionine gene and the proinsulin gene into a bacterial gene in aplasmid vector that is introduced. Proinsulin is then released from thebacterial protein by destruction of the methionine linker, refolded andthe C-peptide removed to yield active insulin.

Many proinsulin variants with modifications to the C-peptide have beenattempted. For example, EP 704,527 discloses the use of any peptidewhich contains at least one N-glycosylation site, preferably-Asn-X-Ser-. Additional C-peptide variants contain from 2 to 35 aminoacids (DK-A-5284/87), or simply a dipeptide of -Lys-Arg- (EP-A-195-691).Other C-peptide variants Other C-peptides variants have been describedas only containing at least one proteolytic cleavage site, preferably-KR-X-KR- (SEQ ID NO:5), -KR-X-M-(SEQ ID NO:6) or -N-X-KR- (SEQ IDNO:7), wherein X is any chain of residues to facilitate cleavage andprocessing by host yeast cells. The advantage of host processing is thatit eliminates or reduces the need for processing the proinsulin throughdigestion and purification steps in order to arrive at the mature form.Other C-peptide modifications include the deletion of residues Arg32 toGlu35, Arg32 to Asp36, Arg32 to Glu35 or Arg32 to Gly60 to render theprecursor susceptible to a site specific protease generated from amutant Pseudomonasfragi (WO 86/01540). Another strategy includes theconnecting molecule -X-Y-, wherein X is a moiety joining the α-aminogroup of A1 and to either the ε-amino group of B-29 or the carboxylgroup of B-30, which is enzymatically or chemically cleavable withoutdisruption to either the A or B insulin chains; and Y is Lys-B30,wherein B30 is Ala, Thr or Ser. (U.S. Pat. No. 4,430,266).

Other expression strategies have deleted the C-peptide entirely, andhave expressed a variant “proinsulin” as a peptide containing ashortened B-chain (B1-29-carboxyl Ala residue deleted) connected via theC-terminal with the N-terminal end of the complete A chain (A1-21).Alternatively, the C-peptide may be from 1-33 residues so long as thereare not two adjacent basic amino acids, for example, -Ser-Lys- or-Ala-Ala-Lys (EP-A-427-296), alternatively -Arg-Arg-Gly-Ser-Lys-Arg-(SEQ ID NO:8) or -Arg-Arg- (EP 055,945), alternatively -Arg- or Lys-Arg-(WO 96/20724).

Various other strategies have been employed to increase the expressionlevels in prokaryotic hosts, including the insertion of amino acidresidues (e.g., Ala, Arg, Gin, Gly, Ile, Leu, Lys, Met, Phe, Ser, Thr,Try or Val) between the methionine and the coding sequence (EP 0 534705). An alternative production scheme involving the production ofGPI-fusion proteins commensurate with deactivation of gas-1 in yeast isdescribed in WO 95/22614, EP 324,274, EP 557,976.

Finally, the art is replete with many mature insulin variants havingantidiabetic activity. For example, E.P. 0 544 466 describes variantshaving residue B3 and A21 independently selected from Ala, Arg, Asn,Cys, Gly, Gin, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp,Tyr and Val in combination with BIO being His, Asp or Glu. U.S. Pat. No.5,514,646 discloses variants which dimerize less readily and aretherefore more rapidly active than the unaltered mature molecule. Forexample, A21 is selected from Ala, Asn, Asp, Gin, Glu, Gly, Thr or Ser;B1 is Phe, Asp or absent; B2 is Val, or absent when B1 is absent; B3 isAsn or Asp; B9 is Ser or Asp; BIO is His or Asn; B28 is any naturallyoccurring amino acid; B29 is Pro, hydroxypro; B30 is Ala, Thr or absent;and X-Y-Z are C-terminal additions to residue B30 wherein Z is —OH,methoxy or ethoxy, X is Arg, Arg-Arg, Lys, Lys-Lys, Arg-Lys, Lys-Arg orabsent and Y is present only when X is present and is Glu or anyfragment of the sequence-Glu-Ala-Glu-Asp-Leu-Gln-Val-Gly-Gln-Val-Glu-Leu-Gly-Gly-Gly-Pro-Gly-Ala-Gly-Ser-Leu-Gln-Pro-Leu-Ala-Leu-Gly-Gly-Ser-Leu-Gln-Lys-Arg(SEQ ID NO:9). WO 95/07931 discloses variants in which residues A21 andB3 are independently any natural amino acid other than Lys, Arg and Cys,B1 is Phe or deleted, B30 is deleted or any natural amino acid otherthan Lys, Arg or Cys having a lipophilic substituent on the α-aminogroup of B29. B30 may also be replaced by a non-codable lipophilic aminoacid having from 10 to 24 carbon atoms having an acyl group with up to 5carbon atoms bound to the α-amino group of B29. EP 0 214 826 describesinsulin variants in which from 1-7 of the following are substituted withthe same or different residues so as to give the mature molecule thesame charge or a greater negative charge at neutral pH than that ofhuman insulin: A9-10, A13, A21, B1-2, B5, B9-10, B12, B14, B16-18, B20,B26-28. RD 365030 (Research Disclosure, September 1994) discloses thesubstitution of Asp for Pro at residue 28 and the deletion of B30 inorder to permit the conversion of proinsulin into the active form by alysyl specific endopeptidase from Achromobacter lyticus instead oftrypsin-activated cleavage. WO 89/10937 discloses insulin variants inwhich one or more Asp or Gin residues (e.g., A5, A15, B4) have beenreplaced by another naturally occurring amino acid. EP 0 375 437discloses insulin variants in which a positively charged amino acid(i.e., Lys or Arg) is substituted at position B28 or alternatively thedeletion of one of residues B24, B25, B26, B27 or B28 resulting in apositive charge at the new B28 residue. U.S. Pat. No. 5,656,722discloses long acting insulin variants in which residue A21 is Gly, Ala,Ser, Thr, Val, Leu, Ile, Asp, Glu, Cys, Met, Arg, Lys, His, Tyr, Phe,Trp, Pro; B1 is absent or is Phe, B10 is any naturally occurring aminoacid (preferably Asn or Gln); B30 is a neutral amino acid (e.g., Ala,Ser, Thr); B31 is a basic organic group having up to 50 carbon atomscomprising from 1-3 basic α-amino acids (e.g., Arg, Lys, Hyl, Orn, Cit,His). The delayed onset is believed to be due to the sparing solubilityat the isoelectric point caused by the basic groups. These moleculebecome active upon enzymatic cleavage of the basic groups (e.g.,trypsin, carboxypeptidase B, esterase). A further variation of thesemolecules is described in U.S. Pat. No. 5,506,302 wherein the basicresidue arginine is positioned at the N-terminal of the A1 glycineresidue. E.P. 0 519 750 discloses insulin variants with Aspsubstitutions at residue B10 in combination with the deletion of B27-30or B28-30, optionally including substitution of residue A21 with Asn,Asp, Glu, Gin, Ala, Gly or Ser, residue B1 with Phe or Asp or residue B3with Asn or Asp.

Finally, the following molecules have been described as havinganti-diabetic activity:

(1) GIVEQ(C)₁(C)₂TSI(C)₁SLYQLENY(C)₃N (SEQ ID NO:10)FVNQHL(C)₂GSHLVEALYLV(C)₃GERGFFYTPKTRREAEDLQVGQVELGGGPGAGSLQPLX (SEQ IDNO:11), wherein the subscripts represent the location of disulfide bondsand X is present or absent, and if present is ALEGSLQ (SEQ ID NO:12) orALEGSLQKR (SEQ ID NO:13)(EP 0 171,886);

(2) EAEDLQVGQVELGGGGAGSLQPLALEGSLQKR-GIVEQ(C)₁(C)₂TSICSLYQLENY(C)₃N (SEQID NO:14)

FVNQHL(C)₂GSHLVEALYLV(C)₃GE-RGFFYTPKT-(RR)_(n), wherein n is 1 (SEQ IDNO:15) or 0 (SEQ ID NO:16)(E.P 171,147), wherein the subscriptsrepresent the location of disulfide bonds; or

(3) ALEGSLQKRGIVEQ(C)₁(C)₂TSI(C)₁SLYQLENY(C)₃N (SEQ ID NO:17)FVNQHL(C)₂GSHLVEALYLV(C)₃GERGFFYTPKTX, wherein the subscripts representthe location of disulfide bonds (EP 171,887), and X is present or absent(SEQ ID NO:18), and if present is -R-R or -RREAEDLQVGQVELGGGPGAGSLQPL(SEQ ID NO:19).

In the presence of ionic zinc (Zn²⁺), natural human insulin associatesto a hexamer with 2 Zn atoms coordinated octahedrally to HB10 of eachmonomer and 3 water molecules. Blundell et al., Adv. Protein Chem. 26:279-402 (1972), Baker et al., Philos. Trans. R. Soc. London B 319,369-456 (1988). The Zn²⁺ binding thereby causes allostericconformational changes.

Phenolic ligands or certain salts are capable of inducing aconformational transition, resulting in the N-terminal 8 amino acids ofthe B-chain converting from and extended conformation to an α-helix.This conformational change allows two Zn atoms to become tetrahedrallycoordinated to HB10 of each monomer and a fourth solvent-accessible siteto be occupied by small anionic ligands, i.e., Cl¹⁻ ion, Brader & Dunn,TIBS 16: 341-345 (1991). The conformational state induced by phenolicligands is referred to as the R state while the pre-conformational formas the T state. Monod et al., J. Mol. Biol. 12: 88-118 (1965). The Rstate is more compact, less flexible, and the Zn is more tightly boundcompared to the T state. Derewenda et al., Nature 338: 594-596 (1989).The conversion of insulin between the T and R allosteric conformationsinvolves the movement of the B(1) alpha carbon a distance greater than30 Å, a very sizeable distance for an allosteric transformation.

The conformational change instigated by the presence of phenolic ligandshas the effect of increasing shelf-life stability. Brange et al., Pharm.Res. 9: 715-726 (1992); Brange et al., Pharm. Res. 9: 727-734 (1992);Brange & Langkjaer, Acta Pharm. Nord. 4: 149-158 (1992). One theoryexplaining why shelf life is increased is through a thermodynamic modeldescribing the degradation of insulin by the equilibrium constant ofunfolding, K_(eq). Brems et al., Protein Engineering 5: 519-525 (1992).This equilibrium constant of [U]/[N] is determined from the reactionN←→U, wherein N is native and U is insulin in unfolded conformation.Since the R state is the conformation more compacted and less flexible,and Zn is more tightly bonded, the R state is also expected to providethe greatest protection from degradation.

The rate of absorption of insulin is directly related to thedissociation constant for self-association. Brange et al., Diabetes Care13: 923-954 (1990). The form of insulin which is actually absorbed isbelieved to be a monomer and the rate of dissociation is therate-limiting step. Binder, C., Artificial Systems for Insulin Delivery,Brunetti et al., Eds, Raven Press, N.Y., 53-57 (1983). Moreover,monomeric insulin analogs are rapidly absorbed and result in a rapidtime action profile. Brange et al., Curr. Opin. Struct. Biol. 1: 934-940(1991); DiMarchi et al., Peptides: Chemistry and Biology, Proceedings ofthe Twelfth American Peptide Symposium, ESCOM, Leiden, pp. 26-28 (1992).Thus the presence of Zn and/or other phenolics has been attempted topostpone the dissociation of the Zn-insulin hexamers and increase shelflife. However, insulin can be induced into a hexamer in the absence ofzinc by substituting GluB13 for GlnB13. Gentley, et al., J. Mol. Biol.228: 1163-1176. Moreover, the hexamer may also be induced in the absenceof insulin by the substitution of an aspartic acid at position 1 of theB chain and optionally glutamine at position B13 (WO 96/04307).

B. Insulin Variants

In addition to the full-length native sequence insulin polypeptidesdescribed herein, it is possible to create insulin variants. Whereaschanges in the formulation may be done to effect the desired changes inactivity, the term “insulin variants” is explicity intended to coverchanges and/or modifications to the polypeptide sequence. Discussion ofmodifications in the formulation appears under section “E.Pharmaceutical compositions and dosages.” Other formulation or sequencechanges in insulin could, in theory, alter the processing and/or theintracellular trafficking of an insulin variant, improve binding to thereceptor and/or protein stability, etc. Such changes could thusultimately improve the bioactivity of an insulin variant relative to thenative molecule.

Variations in the amino acid sequence of insulin or in various domainsof the insulin polypeptide described herein, can be made, for example,using any of the techniques and guidelines for conservative andnon-conservative mutations set forth, for instance, in U.S. Pat. No.5,364,934. Variations may be a substitution, deletion or insertion ofone or more nucleotides encoding the insulin polypeptide that results ina change in the amino acid sequence of the polypeptide as compared withthe native sequence insulin polypeptide. Optionally the variationresults in substitution of at least one amino acid with any other aminoacid in one or more of the domains of the insulin polypeptide. Guidancein determining which amino acid residue may be inserted, substituted ordeleted without adversely affecting the desired activity may be found bycomparing the sequence of the insulin polypeptide with that ofhomologous known protein molecules and minimizing the number of aminoacid sequence changes made in regions of high homology. Amino acidsubstitutions can be the result of replacing one amino acid with anotheramino acid having similar structural and/or chemical properties, such asthe replacement of a leucine with a serine, i.e., conservative aminoacid replacements. Insertions or deletions may optionally be in therange of about 1 to 5 amino acids. The variation allowed may bedetermined by systematically making insertions, deletions orsubstitutions of amino acids in the sequence and testing the resultingvariants for activity exhibited by the full-length or mature nativesequence.

Insulin polypeptide fragments of the polypeptides of the invention arealso within the scope of the invention. Such fragments may be truncatedat the N-terminus or C-terminus, or may lack internal residues, forexample, when compared with a full length native protein. Certainfragments lack amino acid residues that are not essential for a desiredbiological activity of the insulin or insulin variant polypeptide.

Insulin fragments may be prepared by any of a number of conventionaltechniques. Desired peptide fragments may be chemically synthesized. Analternative approach involves generating insulin fragments by enzymaticdigestion, e.g., by treating the protein with an enzyme known to cleaveproteins at sites defined by particular amino acid residues, or bydigesting the DNA with suitable restriction enzymes and using thedesired fragment to generate recombinant protein. Yet another suitabletechnique involves isolating and amplifying a DNA fragment encoding adesired polypeptide fragment, by polymerase chain reaction (PCR).Oligonucleotides that define the desired termini of the DNA fragment areemployed as the 5′ and 3′ primers in the PCR. Preferably, polypeptidefragments share at least one biological and/or immunological activitywith the insulin A-chain and B-chain polypeptide shown in FIGS. 18A& 18B(SEQ ID NO:1-2).

In particular embodiments, conservative substitutions of interest areshown in Table I under the heading of preferred substitutions. If suchsubstitutions result in a change in biological activity, then moresubstantial changes, called exemplary substitutions in Table 6, or asfurther described below in reference to amino acid classes, areintroduced prior to screening the resultant protein products.

TABLE 6 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) val; leu; ile val Arg (R) lys; gln; asn lys Asn (N) gln; his;lys; arg gln Asp (D) glu glu Cys (C) ser ser Gln (Q) asn asn Glu (E) aspasp Gly (G) pro; ala ala His (H) asn; gln; lys; arg arg Ile (I) leu;val; met; ala; phe; leu norleucine Leu (L) norleucine; ile; val; ilemet; ala; phe Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe(F) leu; val; ile; ala; tyr leu Pro (P) ala ala Ser (S) thr thr Thr (T)ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile;leu; met; phe; leu ala; norleucine

Substantial modifications in function or immunological identity of theinvention polypeptide are accomplished by selecting substitutions thatalter the (a) structure of the polypeptide backbone in the area of thesubstitution, for example, as a sheet or helical conformation, (b)charge or hydrophobicity of the molecule at the target site, or (c) bulkof the side chain. Naturally occurring residues are divided into groupsbased on common side-chain properties:

-   (1) hydrophobic: norleucine, met, ala, val, leu, ile;-   (2) neutral hydrophilic: cys, ser, thr;-   (3) acidic: asp, glu;-   (4) basic: asn, gln, his, lys, arg;-   (5) residues that influence chain orientation: gly, pro; and-   (6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Such substituted residues also may beintroduced into the conservative substitution sites or, more preferably,into the remaining (non-conserved) sites.

The variations can be made using methods known in the art such asoligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,and PCR mutagenesis. Site-directed mutagenesis, Carter et al., Nucl.Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487(1987), cassette mutagenesis, Wells et al., Gene, 34:315 (1985),restriction selection mutagenesis, Wells et al., Philos. Trans. R. Soc.London SerA, 317:415 (1986) or other known techniques can be performedon the cloned DNA to produce the variant DNA.

Scanning amino acid analysis can also be employed to identify one ormore amino acids along a contiguous sequence. Among the preferredscanning amino acids are relatively small, neutral amino acids. Suchamino acids include alanine, glycine, serine, and cysteine. Alanine istypically a preferred scanning amino acid among this group because iteliminates the side-chain beyond the beta-carbon and is less likely toalter the main-chain conformation of the variant. Cunningham and Wells,Science, 244: 1081-1085 (1989). Alanine is also typically preferredbecause it is the most common amino acid. Further, it is frequentlyfound in both buried and exposed positions. Creighton, The Proteins,(W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976). Ifalanine substitution does not yield adequate amounts of variant, anisosteric amino acid may be used.

C. Modifications of Insulin and Insulin Variants

Covalent modifications of insulin and/or insulin variants are includedwithin the scope of this invention. One type of covalent modificationincludes reacting targeted amino acid residues of an insulin or insulinvariant polypeptide with an organic derivatizing agent that is capableof reacting with selected side chains or the N- or C-terminal residuesof the molecule. Derivatization with bifunctional agents is useful, forinstance, for crosslinking the molecule to a water-insoluble supportmatrix or surface for use in the method for purifying anti-insulin oranti-insulin variant antibodies, and vice-versa. Commonly usedcrosslinking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with4-azidosalicylic acid, homobifunctional imidoesters, includingdisuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate),bifunctional maleimides such as bis-N-maleimido-1,8-octane and agentssuch as methyl-3-[(p-azidophenyl)dithio]propioimidate.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of theα-amino groups of lysine, arginine, and histidine side chains,acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group. T. E. Creighton, Proteins: Structure and MolecularProperties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)],

Another type of covalent modification of the invention polypeptideincluded within the scope of this invention comprises altering thenative glycosylation pattern of the polypeptide. “Altering the nativeglycosylation pattern” is intended for purposes herein to mean deletingone or more carbohydrate moieties found in native sequence polypeptide(either by removing the underlying glycosylation site or by deleting theglycosylation by chemical and/or enzymatic means), and/or adding one ormore glycosylation sites that are not present in the native sequence. Inaddition, the phrase includes qualitative changes in the glycosylationof the native proteins, involving a change in the nature and proportionsof the various carbohydrate moieties present.

Addition of glycosylation sites to the polypeptide may be accomplishedby altering the amino acid sequence. The alteration may be made, forexample, by the addition of, or substitution by, one or more serine orthreonine residues to the native sequence polypeptide (for O-linkedglycosylation sites). The amino acid sequence may optionally be alteredthrough changes at the DNA level, particularly by mutating the DNAencoding the polypeptide at preselected bases such that codons aregenerated that will translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on thepolypeptide of the invention is by chemical or enzymatic coupling ofglycosides to the polypeptide. Such methods are described in the art,e.g., in WO 87/05330 published 11 Sep. 1987, and in Aplin and Wriston,CRC Crit. Rev. Biochem., pp. 259-306 (1981).

Removal of carbohydrate moieties present on the polypeptide of theinvention may be accomplished chemically or enzymatically or bymutational substitution of codons encoding for amino acid residues thatserve as targets for glycosylation. Chemical deglycosylation techniquesare known in the art and described, for instance, by Hakimuddin, et al.,Arch. Biochem. Biophys., 259:52 (1987) and by Edge et al., Anal.Biochem., 118:131 (1981). Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., Meth. Enzymol.,138:350 (1987).

Another type of covalent modification comprises linking the inventionpolypeptide to one of a variety of nonproteinaceous polymers, e.g.,polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, inthe manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192 or 4,179,337.

The insulin and/or insulin variant polypeptides employable with thepresent invention may also be modified in a way to form a chimericmolecule comprising the invention polypeptide fused to another,heterologous polypeptide or amino acid sequence.

In one embodiment, such a chimeric molecule comprises a fusion of theinvention polypeptide with a tag polypeptide which provides an epitopeto which an anti-tag antibody can selectively bind. The epitope tag isgenerally placed at the amino- or carboxyl-terminus of the polypeptideof the invention. The presence of such epitope-tagged forms of thepolypeptide of the invention can be detected using an antibody againstthe tag polypeptide. Also, provision of the epitope tag enables thepolypeptide of the invention to be readily purified by affinitypurification using an anti-tag antibody or another type of affinitymatrix that binds to the epitope tag. Various tag polypeptides and theirrespective antibodies are well known in the art. Examples includepoly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags;the flu HA tag polypeptide and its antibody 12CA5, Field et al., Mol.Cell. Biol., 8:2159-2165 (1988); the c-myc tag and the 8F9, 3C7, 6E10,G4, B7 and 9E10 antibodies thereto, Evan et al., Molecular and CellularBiology, 5:3610-3616 (1985); and the Herpes Simplex virus glycoprotein D(gD) tag and its antibody, Paborsky et al., Protein Engineering,3(6):547-553 (1990). Other tag polypeptides include the Flag-peptide,Hopp et al., BioTechnology, 6:1204-1210 (1988); the KT3 epitope peptide,Martin et al., Science, 255:192-194 (1992); an α-tubulin epitopepeptide, Skinner et al., J. Biol. Chem., 266:15163-15166 (1991); and theT7 gene 10 protein peptide tag, Lutz-Freyermuth et al., Proc. Natl.Acad. Sci. USA, 87:6393-6397 (1990).

In an alternative embodiment, the chimeric molecule may comprise afusion of the polypeptide of the invention with an immunoglobulin or aparticular region of an immunoglobulin. For a bivalent form of thechimeric molecule (also referred to as an “immunoadhesin”), such afusion could be to the Fc region of an IgG molecule. The Ig fusionspreferably include the substitution of a soluble (transmembrane domaindeleted or inactivated) form of an invention polypeptide in place of atleast one variable region within an Ig molecule. In a particularlypreferred embodiment, the immunoglobulin fusion includes the hinge, CH2and CH3, or the hinge, CH1, CH2 and CH3 regions of an IgG1 molecule. Forthe production of immunoglobulin fusions see also U.S. Pat. No.5,428,130 issued Jun. 27, 1995.

D. Preparation of Insulin or Insulin Variant Polypeptides

The description below relates primarily to production of insulin andinsulin variant polypeptide by culturing cells transformed ortransfected with a vector containing insulin or insulin variant nucleicacid. Alternative methods, which are well known in the art, could alsobe employed to prepare such polypeptides. For instance, the sequence, orportions thereof, may be produced by direct peptide synthesis usingsolid-phase techniques (see, e.g., Stewart et al., Solid-Phase PeptideSynthesis, W.H. Freeman Co., San Francisco, Calif. (1969); Merrifield,J. Am. Chem. Soc. 85: 2149-2154 (1963)). In vitro protein synthesis maybe performed using manual techniques or by automation. Automatedsynthesis may be accomplished, for instance, using an Applied BiosystemsPeptide Synthesizer (Foster City, Calif.) using the manufacturer'sinstructions. Automated synthesis may be accomplished, for instance,using an Applied Biosystems Peptide Synthesizer (Foster City, Calif.)using the manufacturer's instructions. Various portions of thepolypeptide may be chemically synthesized separately and combined usingchemical or enzymatic methods to produce the full-length insulin orinsulin variant polypeptides.

1. Isolation of DNA Encoding the Polypeptide of the Invention

DNA encoding insulin or insulin variant may be obtained from a cDNAlibrary prepared from tissue believed to express the insulin or insulinvariant mRNA and to express it at a detectable level. Accordingly, humaninsulin or insulin variant DNA can be conveniently obtained from a cDNAlibrary prepared from human tissue. The insulin or insulinvariant-encoding gene may also be obtained from a genomic library or byoligonucleotide synthesis.

Libraries can be screened with probes (such as antibodies to thepolypeptide of the invention or oligonucleotides of at least about 20-80bases) designed to identify the gene of interest or the protein encodedby it. Screening the cDNA or genomic library with the selected probe maybe conducted using standard procedures, such as described in Sambrook etal., Molecular Cloning: A Laboratory Manual (New York: Cold SpringHarbor Laboratory Press, 1989). An alternative means to isolate the geneencoding the insulin and/or insulin variant polypeptides is to use PCRmethodology. Sambrook et al., supra; Dieffenbach et al., PCR Primer: ALaboratory Manual (Cold Spring Harbor Laboratory Press, 1995).

The Examples below describe techniques for screening a cDNA library. Theoligonucleotide sequences selected as probes should be of sufficientlength and sufficiently unambiguous that false positives are minimized.The oligonucleotide is preferably labeled such that it can be detectedupon hybridization to DNA in the library being screened. Methods oflabeling are well known in the art, and include the use of radiolabelslike ³²P-labeled ATP, biotinylation or enzyme labeling. Hybridizationconditions, including moderate stringency and high stringency, areprovided in Sambrook et al., supra.

Sequences identified in such library screening methods can be comparedand aligned to other known sequences deposited and available in publicdatabases such as GenBank or other private sequence databases. Sequenceidentity (at either the amino acid or nucleotide level) within definedregions of the molecule or across the full-length sequence can bedetermined through sequence alignment using computer software programssuch as ALIGN, DNAstar, and INHERIT which employ various algorithms tomeasure homology.

Nucleic acid having protein coding sequence may be obtained by screeningselected cDNA or genomic libraries using the deduced amino acid sequencedisclosed herein for the first time, and, if necessary, usingconventional primer extension procedures as described in Sambrook etal., supra, to detect precursors and processing intermediates of mRNAthat may not have been reverse-transcribed into cDNA.

2. Selection and Transformation of Host Cells

Host cells are transfected or transformed with expression or cloningvectors described herein for insulin or insulin variant production andcultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences. General principles, protocols, andpractical techniques for maximizing the productivity of cell culturescan be found in Mammalian Cell Biotechnology: A Practical Approach, M.Butler, ed. (IRL Press, 1991) and Sambrook et al., supra.

Methods of transfection include CaCl₂, CaPO₄, liposome-mediated andelectroporation. Depending on the host cell used, transformation isperformed using standard techniques appropriate to such cells. Thecalcium treatment employing calcium chloride, as described in Sambrooket al., supra, or electroporation is generally used for prokaryotes orother cells that contain substantial cell-wall barriers. Infection withAgrobacterium tumefaciens is used for transformation of certain plantcells, as described by Shaw et al., Gene, 23:315 (1983) and WO 89/05859published 29 Jun. 1989. For mammalian cells without such cell walls, thecalcium phosphate precipitation method of Graham and van der Eb,Virology, 52:456-457 (1978) can be employed. General aspects ofmammalian cell host system transformations have been described in U.S.Pat. No. 4,399,216. Transformations into yeast are typically carried outaccording to the method of Van Solingen et al., J. Bact., 130: 946(1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979).However, other methods for introducing DNA into cells, such as bynuclear microinjection, electroporation, bacterial protoplast fusionwith intact cells, polycations, e.g., polybrene, polyornithine, or useof recombinant viral vectors, may also be used. For various techniquesfor transforming mammalian cells, see Keown et al., Methods inEnzymology, 185:527-537 (1990) and Mansour et al., Nature, 336:348-352(1988).

Suitable host cells for cloning or expressing the DNA in the vectorsherein include prokaryote, yeast, or higher eukaryote cells. Suitableprokaryotes include but are not limited to eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as E. coli. Various E. coli strains are publiclyavailable, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776(ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5 772 (ATCC53,635). Other suitable prokaryotic host cells includeEnterobacteriaceae such as Escherichia, e.g., E. coli K12 strain MM294(ATCC 31,446); E. coli X1776 (ATCC 31,537); E. coli strain W3110 (ATCC27,325) and K5 772 (ATCC 53,635), Enterobacter, Erwinia, Klebsiella,Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g.,Serratia marcescans, and Shigella, as well as Bacilli such as B.subtilis and B. licheniformis (e.g., B. licheniformis 41P disclosed inDD266,710 published 12 Apr. 1989), Pseudomonas such as P. aeruginosa,and Streptomyces. These examples are illustrative rather than limiting.Strain W3110 is one particularly preferred host or parent host becauseit is a common host strain for recombinant DNA product fermentations.Preferably, the host cell secretes minimal amounts of proteolyticenzymes. For example, strain W3110 may readily modified to turn of itsendogenous genes in favor of expression of the heterologous sequence.For example, E. coli W3110 strain 1A2, which has the complete genotypetonA; E. coli W3110 strain 9E4, which has the complete genotype tonAptr3; E coli W3110 strain 27C7 (ATCC 55,244), which has the completegenotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT kan^(r) ; E. coliW3110 strain 37D6, which has the complete genotype tonA ptr3 phoA E15(argF-lac)169 degP ompT rbs7 ilvG kan; E. coli W3110 strain 40B4, whichis strain 37D6 with a non-kanamycin resistant degP deletion mutation;and an E. coli strain having mutant periplasmic protease disclosed inU.S. Pat. No. 4,946,83 issued 7 Aug. 1990.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for insulin orinsulin variant encoding vectors. Saccharomyces cerevisiae is a commonlyused lower eukaryotic host microorganism. Others includeSchizosaccharomyces pombe (Beach and Nurse, Nature 290: 140 (1981); EP139,383 published 2 May 1985); Kluveromyces hosts (U.S. Pat. No.4,943,529; Fleer et al., Bio/Technology 9: 968-975 (1991)) such as,e.g., K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al., J.Bacteriol. 154(2): 737 (1983); K. fragilis (ATCC 12,424), K. bulgaricus(ATCC 16,045), K. wicheramii (ATCC 24,178), K. waltii (ATCC 56,500), K.drosophilarum (ATCC 36,906); Van den Berg et al., Bio/Technology 8: 135(1990)), K. thermotolerans, and K. marxianus; yarrowia (EP 402,226);Pichia pastoris (EP 183,070); Sreekrishna et al., J. Basic Microbiol.28: 265-278 (1988); Candida; Trichoderma reesia (EP 244,234); Neurosporacrassa (Case et al., Proc. Natl. Acad. Sci. USA, 76:5259-5263 (1979);Schwanniomyces such as Schwanniomyces occidentalis (EP 394,538 published31 Oct. 1990); and filamentous fungi such as, e.g., Neurospora,Penicillium, Tolypocladium (WO 91/00357 published 10 Jan. 1991), andAspergillus hosts such as A. nidulans (Ballance et al, Biochem. Biophys.Res. Commun. 112: 284-289(1983); Tilburn et al., Gene, 26: 205-221(1983); Yelton et al., Proc. Natl. Acad. Sci. USA 81: 1470-1474 (1984))and A. niger (Kelly and Hynes, EMBO J. 4: 475-479 (1985)). Methylotropicyeasts are suitable herein and include, but are not limited to, yeastcapable of growth on methanol selected from the genera consisting ofHansenula, Cadida, Kloeckera, Pichia, Saccharomyces, Torulopsis, andRhodotorula. A list of specific species that are exemplary of this classof yeasts may be found in C. Anthony, The Biochemistry of Methylotrophs269 (1982).

Suitable host cells for the expression of glycosylated insulin andinsulin variant polypeptides can be derived from multicellularorganisms. Examples of invertebrate cells include insect cells such asDrosophila S2 and Spodoptera Sf9 and high five, as well as plant cells.Examples of useful mammalian host cell lines include Chinese hamsterovary (CHO) and COS cells. More specific examples include monkey kidneyCV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonickidney line (293 or 293 cells subcloned for growth in suspensionculture, Graham et al., J. Gen Virol., 36:59 (1977)); Chinese hamsterovary cells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA,77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.,23:243-251 (1980)); human lung cells (W138, ATCC CCL 75); human livercells (Hep G2, HB 8065); and mouse mammary tumor (MMT 060562, ATCCCCL51). The selection of the appropriate host cell is deemed to bewithin the skill in the art.

3. Selection and Use of a Replicable Vector

The nucleic acid (e.g., cDNA or genomic DNA) encoding insulin or insulinvariants may be inserted into a replicable vector for cloning(amplification of the DNA) or for expression. Various vectors arepublicly available. The vector may, for example, be in the form of aplasmid, cosmid, viral particle, phagemid or phage. The appropriatenucleic acid sequence may be inserted into the vector by a variety ofprocedures. In general, DNA is inserted into an appropriate restrictionendonuclease site(s) using techniques known in the art. Vectorcomponents generally include, but are not limited to, one or more of asignal sequence, an origin of replication, one or more marker genes, anenhancer element, a promoter, and a transcription termination sequence.Construction of suitable vectors containing one or more of thesecomponents employs standard ligation techniques which are known to theskilled artisan.

The insulin or insulin variant polypeptide may be produced recombinantlynot only directly, but also as a fusion polypeptide with a heterologouspolypeptide, which may be a signal sequence or other polypeptide havinga specific cleavage site at the N-terminus of the mature protein orpolypeptide. In general, the signal sequence may be a component of thevector, or it may be a part of the insulin or insulin variant-encodingDNA that is inserted into the vector. The signal sequence may be aprokaryotic signal sequence selected, for example, from the group of thealkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin IIleaders. For yeast secretion the signal sequence may be, e.g., the yeastinvertase leader, alpha factor leader (including Saccharomyces andKluyveromyces α-factor leaders, the latter described in U.S. Pat. No.5,010,182), or acid phosphatase leader, the C. albicans glucoamylaseleader (EP 362,179 published 4 Apr. 1990), or the signal described in WO90/13646 published 15 Nov. 1990. In mammalian cell expression, mammaliansignal sequences may be used to direct secretion of the protein, such assignal sequences from secreted polypeptides of the same or relatedspecies, as well as viral secretory leaders.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells. Suchsequences are well known for a variety of bacteria, yeast, and viruses.The origin of replication from the plasmid pBR322 is suitable for mostGram-negative bacteria, the 2μ plasmid origin is suitable for yeast, andvarious viral origins (SV40, polyoma, adenovirus, VSV or BPV) are usefulfor cloning vectors in mammalian cells.

Expression and cloning vectors will typically contain a selection gene,also termed a selectable marker. Typical selection genes encode proteinsthat (a) confer resistance to antibiotics or other toxins, e.g.,ampicillin, neomycin, methotrexate, or tetracycline, (b) complementauxotrophic deficiencies, or (c) supply critical nutrients not availablefrom complex media, e.g., the gene encoding D-alanine racemase forBacilli.

An example of suitable selectable markers for mammalian cells are thosethat enable the identification of cells competent to take up the nucleicacid encoding the polypeptide of the invention, such as DHFR orthymidine kinase. An appropriate host cell when wild-type DHFR isemployed is the CHO cell line deficient in DHFR activity, prepared andpropagated as described by Urlaub et al., Proc. Natl. Acad. Sci. USA,77:4216 (1980). A suitable selection gene for use in yeast is the trp1gene present in the yeast plasmid YRp7. Stinchcomb et al., Nature,282:39 (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et al,Gene, 10:157 (1980). The trp1 gene provides a selection marker for amutant strain of yeast lacking the ability to grow in tryptophan, forexample, ATCC No. 44076 or PEP4-1. Jones, Genetics, 85:12 (1977).

Expression and cloning vectors usually contain a promoter operablylinked to the insulin-encoding nucleic acid sequence to direct mRNAsynthesis. Promoters recognized by a variety of potential host cells arewell known. Promoters suitable for use with prokaryotic hosts includethe β-lactamase and lactose promoter systems; Chang et al., Nature,275:615 (1978); Goeddel et al., Nature, 281:544 (1979); alkalinephosphatase, a tryptophan (trp) promoter system, Goeddel, Nucleic AcidsRes., 8:4057 (1980); EP 36,776; and hybrid promoters such as the tacpromoter, deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983).Promoters for use in bacterial systems also will contain aShine-Dalgarno (S.D.) sequence operably linked to the DNA encoding theinsulin or insulin variant polypeptide of the invention.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase, Hitzeman et al., J.Biol. Chem., 255:2073 (1980) or other glycolytic enzymes, Hess et al.,J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900(1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657.

Expression from vectors in mammalian host cells is controlled, forexample, by promoters obtained from the genomes of viruses such aspolyoma virus, fowlpox virus (UK 2,211,504 published 5 Jul. 1989),adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcomavirus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus40 (SV40), from heterologous mammalian promoters, e.g., the actinpromoter or an immunoglobulin promoter, and from heat-shock promoters,provided such promoters are compatible with the host cell systems.

Transcription of a DNA encoding the polypeptides employable with theinvention by higher eukaryotes may be increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp, that act on a promoter to increase itstranscription. Many enhancer sequences are now known from mammaliangenes (globin, elastase, albumin, α-fetoprotein, and insulin).Typically, however, one will use an enhancer from a eukaryotic cellvirus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. The enhancer may be spliced into thevector at a position 5′ or 3′ to the insulin or insulin variant codingsequence, but is preferably located at a site 5′ from the promoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding the polypeptides of the invention.

Still other methods, vectors, and host cells suitable for adaptation tothe synthesis of the polypeptides of the invention in recombinantvertebrate cell culture are described in Gething et al., Nature,293:620-625 (1981); Mantei et al., Nature, 281:40-46 (1979); EP 117,060;and EP 117,058.

4. Detecting Gene Amplification/Expression

Gene amplification or expression may be measured in a sample directly,for example, by conventional Southern blotting or Northern blotting orRT-PCR (Taqman) to quantitate the transcription of mRNA, Thomas, Proc.Natl. Acad. Sci. USA, 77:5201-5205 (1980), dot blotting (DNA or RNAanalysis), or in situ hybridization, using an appropriately labeledprobe, based on the sequences provided herein.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of cells or tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. Antibodies useful forimmunohistochemical staining and/or assay of sample fluids may be eithermonoclonal or polyclonal, and may be prepared in any mammal.Conveniently, the antibodies may be prepared against a native sequenceinsulin or insulin variant polypeptide or against a synthetic peptidebased on the DNA sequences provided herein or against exogenous sequencefused to insulin or insulin variant DNA encoding the polypeptide of theinvention and encoding a specific antibody epitope.

5. Purification of Polypeptide

Forms of the polypeptides employable with the present invention may berecovered from culture medium or from host cell lysates. Ifmembrane-bound, they can be released from the membrane using a suitabledetergent solution (e.g. Triton®-X 100) or by enzymatic cleavage. Cellsemployed in expression of the polypeptide employable with the inventioncan be disrupted by various physical or chemical means, such asfreeze-thaw cycling, sonication, mechanical disruption, or cell lysingagents.

It may be desired to purify insulin or insulin. The following proceduresare exemplary of suitable purification procedures: by fractionation onan ion-exchange column; ethanol precipitation; reverse phase HPLC;chromatography on silica or on a cation-exchange resin such as DEAE;chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gelfiltration using, for example, Sephadex G-75; protein A Sepharosecolumns to remove contaminants such as IgG; and metal chelating columnsto bind epitope-tagged forms of the polypeptide of the invention.Various methods of protein purification may be employed and such methodsare known in the art and described for example in Deutscher, Methods inEnzymology, 182 (1990); Scopes, Protein Purification: Principles andPractice, Springer-Verlag, New York (1982). The purification step(s)selected will depend, for example, on the nature of the productionprocess used and the particular insulin or insulin variant produced.

6. Tissue Distribution

The location of tissues expressing the polypeptides employable with theinvention can be identified by determining mRNA expression in varioushuman tissues. Such data may help determine which tissues are mostlikely to be affected by the stimulating and inhibiting activities ofthe polypeptides of the invention. Tissue which expresses and respondsto insulin could in theory be used in the activity blocking assaysdiscussed below.

As noted before, gene expression in various tissues may be measured byconventional Northern blotting, RT-PCR (Taqman), Thomas, Proc. Natl.Acad. Sci. USA, 77:5201-5205 [1980], dot blotting, or in situhybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, gene expression can bemeasured by immunological methods, such as immunohistochemical stainingof tissue sections and assay of cell culture or body fluids, asdescribed above under 4. Detecting Gene Amplification/Expression.

E. Pharmaceutical Compositions and Dosages

The insulin and insulin variant polypeptides employable with the methodsof the invention can be administered for the treatment of cartilaginousdisorders in the form of pharmaceutical compositions. Additionally,lipofections or liposomes can also be used to deliver the insulin orinsulin variant into cells and the target area.

Therapeutic formulations of the active molecules employable with theinvention are prepared for storage by mixing the active molecule havingthe desired degree of purity with optional pharmaceutically acceptablecarriers, excipients or stabilizers (Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. [1980]). Such therapeutic formulations can bein the form of lyophilized formulations or aqueous solutions. Acceptablecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations employed, and include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyidimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose,dextrins, or hyaluronan; chelating agents such as EDTA; sugars such assucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions suchas sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN®, PLURONICS® or polyethylene glycol (PEG).

In order for the formulations to be used in vivo administration, theymust be sterile. The formulation may be readily rendered sterile byfiltration through sterile filtration membranes, prior to or followinglyophilization and reconstitution. The therapeutic compositions hereingenerally are placed into a container having a sterile access port, forexample, an intravenous solution bag or vial having a stopper pierceableby a hypodermic injection needle.

The formulations used herein may also contain more than one activecompound as necessary for the particular indication being treated,preferably those with complementary activities that do not adverselyaffect each other. Alternatively, or in addition, the composition maycomprise a cytotoxic agent, cytokine or growth inhibitory agent. Suchmolecules are present in combinations and amounts that are effective forthe intended purpose.

The route of administration is in accordance with known methods, e.g.,injection or infusion by intravenous, intraperitoneal, intramuscular,intraarterial, intralesional or intraarticular routes, topicaladministration, by sustained release or extended-release means.Optionally the active compound or formulation is injected directly intothe afflicted cartilaginous region or articular joint.

The insulin or insulin variant molecules by also be prepared byentrapping in microcapsules prepared, for example by coacervationtechniques or by interfacial polymerization, for example,hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacrylate) microcapsules, respectively. Such preparationscan be administered in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, 16th Edition (or newer), Osol A.Ed. (1980).

Where sustained-release or extended-release administration of theinsulin or insulin variant polypeptides is desired in a formulation withrelease characteristics suitable for the treatment of any disease ordisorder requiring administration of such polypeptides,microencapsulation is contemplated. Microencapsulation of recombinantproteins for sustained release has been successfully performed withhuman growth hormone (rhGH), interferon-α, -β, -γ (rhIFN-α, -β, -γ),interleukin-2, and MN rgp120. Johnson et al., Nat. Med. 2: 795-799(1996); Yasuda, Biomed. Ther. 27: 1221-1223 (1993); Hora et al.,Bio/Technology 8: 755-758 (1990); Cleland, “Design and Production ofSingle Immunization Vaccines Using Polylactide Polyglycolide MicrosphereSystems” in Vaccine Design: The Subunit and Adjuvant Approach, Powelland Newman, eds., (Plenum Press: New York, 1995), pp. 439-462; WO97/03692, WO 96/40072, WO 96/07399 and U.S. Pat. No. 5,654,010.

Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing theactive molecule, which matrices are in the form of shaped articles, e.g.films, or microcapsules. Examples of sustained-release matrices includeone or more polyanhydrides (e.g., U.S. Pat. Nos. 4,891,225; 4,767,628),polyesters such as polyglycolides, polylactides andpolylactide-co-glycolides (e.g., U.S. Pat. Nos. 3,773,919; 4,767,628;4,530,840; Kulkarni et al., Arch. Surg. 93: 839 (1966)), polyamino acidssuch as polylysine, polymers and copolymers of polyethylene oxide,polyethylene oxide acrylates, polyacrylates, ethylene-vinyl acetates,polyamides, polyurethanes, polyorthoesters, polyacetylnitriles,polyphosphazenes, and polyester hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), cellulose,acyl substituted cellulose acetates, non-degradable polyurethanes,polystyrenes, polyvinyl chloride, polyvinyl fluoride,poly(vinylimidazole), chlorosulphonated polyolefins, polyethylene oxide,copolymers of L-glutamic acid and γ-ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT® (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release proteins for shorter timeperiods. Additional non-biodegradable polymers which may be employed arepolyethylene, polyvinyl pyrrolidone, ethylene vinylacetate, polyethyleneglycol, cellulose acetate butyrate and cellulose acetate propionate.

Alternatively, sustained release formulations may be composed ofdegradable biological materials. Biodegradable polymers are attractivedrug formulations because of their biocompatibility, high responsibilityfor specific degradation, and ease of incorporating the active drug intothe biological matrix. For example, hyaluronic acid (HA) may becrosslinked and used as a swellable polymeric delivery vehicle forbiological materials. U.S. Pat. No. 4,957,744; Valle et al., Polym.Mater. Sci. Eng. 62: 731-735 (1991). HA polymer grafted withpolyethylene glycol has also been prepared as an improved deliverymatrix which reduced both undesired drug leakage and the denaturingassociated with long term storage at physiological conditions. Kazuteru,M., J. Controlled Release 59:77-86 (1999). Additional biodegradablepolymers which may be used are poly(caprolactone), polyanhydrides,polyamino acids, polyorthoesters, polycyanoacrylates,poly(phosphazines), poly(phosphodiesters), polyesteramides,polydioxanones, polyacetals, polyketals, polycarbonates,polyorthocarbonates, degradable and nontoxic polyurethanes,polyhydroxylbutyrates, polyhydroxyvalerates, polyalkylene oxalates,polyalkylene succinates, poly(malic acid), chitin and chitosan.

Alternatively, biodegradable hydrogels may be used as controlled releasedelivery vehicles for biological materials and drugs. Through theappropriate choice of macromers, membranes can be produced with a rangeof permeability, pore sizes and degradation rates suitable for a widevariety of biomolecules.

Alternatively, sustained-release delivery systems for biologicalmaterials and drugs can be composed of dispersions. Dispersions mayfurther be classified as either suspensions or emulsions. In the contextof delivery vehicles for biological materials, suspensions are a mixtureof very small solid particles which are dispersed (more or lessuniformly) in a liquid medium. The solid particles of a suspension canrange in size from a few nanometers to hundreds of microns, and includemicrospheres, microcapsules and nanospheres. Emulsions, on the otherhand, are a mixture of two or more immiscible liquids held in suspensionby small quantities of emulsifiers. Emulsifiers form an interfacial filmbetween the immiscible liquids and are also known as surfactants ordetergents. Emulsion formulations can be both oil in water (o/w) whereinwater is in a continuous phase while the oil or fat is dispersed, aswell as water in oil (w/o), wherein the oil is in a continuous phasewhile the water is dispersed. One example of a suitablesustained-release formulation is disclosed in WO 97/25563. Additionally,emulsions for use with biological materials include multiple emulsions,microemulsions, microdroplets and liposomes. Microdroplets areunilamellar phospholipid vesicles that consist of a spherical lipidlayer with an oil phase inside. E.g., U.S. Pats. No. 4,622,219 and4,725,442. Liposomes are phospholipid vesicles prepared by mixingwater-insoluble polar lipids with an aqueous solution.

Alternatively, the sustained-release formulations of insulin or insulinvariant polypeptides may be developed using poly-lactic-coglycolic acid(PLGA), a polymer exhibiting a strong degree of biocompatibility and awide range of biodegradable properties. The degradation products ofPLGA, lactic and glycolic acids, are cleared quickly from the humanbody. Moreover, the degradability of this polymer can be adjusted frommonths to years depending on its molecular weight and composition. Forfurther information see Lewis, “Controlled Release of Bioactive Agentsfrom Lactide/Glycolide polymer,” in Biogradable Polymers as DrugDelivery Systems M. Chasin and R. Langeer, editors (Marcel Dekker: NewYork, 1990), pp. 141.

When encapsulated polypeptides remain in the body for a long time, theymay denature or aggregate as a result of exposure to moisture at 37° C.,resulting in a loss of biological activity and possible changes inimmunogenicity. Rational strategies can be devised for stabilizationdepending on the mechanism involved. For example, if the aggregationmechanism is discovered to be intermolecular S—S bond formation throughthio-disulfide interchange, stabilization may be achieved by modifyingsulfhydryl residues, lyophilizing from acidic solutions, controllingmoisture content, using appropriate additives, and developing specificpolymer matrix compositions.

The encapsulated polypeptides or polypeptides in extended-releaseformulation may be imparted by formulating the polypeptide with a“water-soluble polyvalent metal salts” which are non-toxic at therelease concentration and temperature. Exemplary “polyvalent metals”include the following cations: Ca²⁺, Mg²⁺, Zn²⁺, Fe²⁺, Fe³⁺, Cu²⁺, Sn²⁺,Sn⁴⁺, Al²⁺ and Al³⁺. Exemplary anions which from water soluble saltswith the above polyvalent metal cations include those formed byinorganic acids and/or organic acids. Such water-soluble salts have asolubility in water (at 20° C.) of at least about 20 mg/ml,alternatively 100 mg/ml, alternatively 200 mg/ml.

Suitable inorganic acids that can be used to form the “water solublepolyvalent metal salts” include hydrochloric, sulfuric, nitric,thiocyanic and phosphoric acid. Suitable organic acids that can be usedinclude aliphatic carboxylic acid and aromatic acids. Aliphatic acidswithin this definition may be defined as saturated or unsaturated C₂₋₉carboxylic acids (e.g., aliphatic mono-, di- and tri-carboxylic acids).For example, exemplary monocarboxylic acids within this definitioninclude the saturated C₂₋₉ monocarboxylic acids acetic, proprionic,butyric, valeric, caproic, enanthic, caprylic pelargonic and capryonic,and the unsaturated C₂₋₉ monocarboxylic acids acrylic, propriolicmethacrylic, crotonic and isocrotonic acids. Exemplary dicarboxylicacids include the saturated C₂₋₉ dicarboxylic acids malonic, succinic,glutaric, adipic and pimelic, while unsaturated C₂₋₉ dicarboxylic acidsinclude maleic, fumaric, citraconic and mesaconic acids. Exemplarytricarboxylic acids include the saturated C₂₋₉ tricarboxylic acidstricarballylic and 1,2,3-butanetricarboxylic acid. Additionally, thecarboxylic acids of this definition may also contain one or two hydroxylgroups to form hydroxy carboxylic acids. Exemplary hydroxy carboxylicacids include glycolic, lactic, glyceric, tartronic, malic, tartaric andcitric acid. Aromatic acids within this definition include benzoic andsalicylic acid.

Commonly employed water soluble polyvalent metal salts which may be usedto help stabilize the encapsulated polypeptides of this inventioninclude, for example: (1) the inorganic acid metal salts of halides(e.g., zinc chloride, calcium chloride), sulfates, nitrates, phosphatesand thiocyanates; (2) the aliphatic carboxylic acid metal salts calciumacetate, zinc acetate, calcium proprionate, zinc glycolate, calciumlactate, zinc lactate and zinc tartrate; and (3) the aromatic carboxylicacid metal salts of benzoates (e.g., zinc benzoate) and salicylates.

Dosages and desired drug concentrations of pharmaceutical compositionsemployable with the present invention may vary depending on theparticular use envisioned. The determination of the appropriate dosageor route of administration is well within the skill of an ordinaryphysician. Animal experiments provide reliable guidance for thedetermination of effective doses for human therapy. Interspecies scalingof effective doses can be performed following the principles laid downby Mordenti, J. and Chappell, W. “The use of interspecies scaling intoxicokinetics” in Toxicokinetics and New Drug Development, Yacobi etal., Eds., Pergamon Press, New York 1989, pp. 42-96.

When in vivo administration of insulin or insulin variant polypeptidesare employed, normal dosage amounts may vary from about 10 ng/kg up toabout 100 mg/kg of mammal body weight or more per day, preferably about1 μg/kg/day to 10 mg/kg/day, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature; see, for example, U.S. Pat. Nos. 4,657,760; 5,206,344 or5,225,212. It is anticipated that different formulations will beeffective for different treatments and different disorders, and thatadministration intended to treat a specific organ or tissue, maynecessitate delivery in a manner different from that to another organ ortissue.

F. Methods of Treatment

It is contemplated that the polypeptides, antibodies and other activecompounds of the present invention may be used to treat variouscartilaginous disorders. Exemplary conditions or disorders to be treatedwith the polypeptides of the invention, include, but are not limited tosystemic lupus erythematosis, rheumatoid arthritis, juvenile chronicarthritis, osteoarthritis, spondyloarthropathies, systemic sclerosis(scleroderma), idiopathic inflammatory myopathies (dermatomyositis,polymyositis), Sjögren's syndrome, systemic vasculitis, sarcoidosis,autoimmune hemolytic anemia (immune pancytopenia, paroxysmal nocturnalhemoglobinuria), autoimmune thrombocytopenia (idiopathicthrombocytopenic purpura, immune-mediated thrombocytopenia), thyroiditis(Grave's disease, Hashimoto's thyroiditis, juvenile lymphocyticthyroiditis, atrophic thyroiditis), diabetes mellitus, immune-mediatedrenal disease (glomerulonephritis, tubulointerstitial nephritis),demyelinating diseases of the central and peripheral nervous systemssuch as multiple sclerosis, idiopathic demyelinating polyneuropathy orGuillain-Barré syndrome, and chronic inflammatory demyelinatingpolyneuropathy, hepatobiliary diseases such as infectious hepatitis(hepatitis A, B, C, D, E and other non-hepatotropic viruses), autoimmunechronic active hepatitis, primary biliary cirrhosis, granulomatoushepatitis, and sclerosing cholangitis, inflammatory bowel disease(ulcerative colitis: Crohn's disease), gluten-sensitive enteropathy, andWhipple's disease, autoimmune or immune-mediated skin diseases includingbullous skin diseases, erythema multiforme and contact dermatitis,psoriasis, allergic diseases such as asthma, allergic rhinitis, atopicdermatitis, food hypersensitivity and urticaria, immunologic diseases ofthe lung such as eosinophilic pneumonias, idiopathic pulmonary fibrosisand hypersensitivity pneumonitis, transplantation associated diseasesincluding graft rejection and graft-versus-host-disease.

In systemic lupus erythematosus, the central mediator of disease is theproduction of auto-reactive antibodies to self proteins/tissues and thesubsequent generation of immune-mediated inflammation. These antibodieseither directly or indirectly mediate tissue injury. Although Tlymphocytes have not been shown to be directly involved in tissuedamage, T lymphocytes are required for the development of auto-reactiveantibodies. The genesis of the disease is thus T lymphocyte dependent.Multiple organs and systems are affected clinically including kidney,lung, musculoskeletal system, mucocutaneous, eye, central nervoussystem, cardiovascular system, gastrointestinal tract, bone marrow andblood.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune inflammatorydisease that affects the synovial membrane of multiple joints and whichresults in injury to the articular cartilage. The pathogenesis is Tlymphocyte dependent and is associated with the production of rheumatoidfactors, auto-antibodies directed against endogenous proteins, with theresultant formation of immune complexes that attain high levels in jointfluid and blood. These complexes may induce infiltration by lymphocytes,monocytes, and neutrophils into the synovial compartment. Tissuesaffected are primarily the joints, often in symmetrical pattern.However, disease outside the joints occurs in two major forms. In oneform, typical lesions are pulmonary fibrosis, vasculitis, and cutaneousulcers. The second form is the so-called Felty's syndrome which occurslate in the RA disease course, sometimes after joint disease has becomequiescent, and involves the presence of neutropenia, thrombocytopeniaand splenomegaly. This can be accompanied by vasculitis in multipleorgans and occurrence of infarcts, skin ulcers and gangrene. Patientsoften also develop rheumatoid nodules in the subcutis tissue overlyingaffected joints; in late stages, the nodules have necrotic centerssurrounded by a mixed inflammatory cellular infiltrate. Othermanifestations which can occur in RA include: pericarditis, pleuritis,coronary arteritis, intestitial pneumonitis with pulmonary fibrosis,keratoconjunctivitis sicca, and rheumatoid nodules.

Juvenile chronic arthritis is a chronic idiopathic inflammatory diseasewhich begins often at less than 16 years of age and which has somesimilarities to RA. Some patients which are rheumatoid factor positiveare classified as juvenile rheumatoid arthritis. The disease issub-classified into three major categories: pauciarticular,polyarticular, and systemic. The arthritis can be severe and leads tojoint ankylosis and retarded growth. Other manifestations can includechronic anterior uveitis and systemic amyloidosis.

Spondyloarthropathies are a group of disorders with some common clinicalfeatures and the common association with the expression of HLA-B27 geneproduct. The disorders include: ankylosing sponylitis, Reiter's syndrome(reactive arthritis), arthritis associated with inflammatory boweldisease, spondylitis associated with psoriasis, juvenile onsetspondyloarthropathy and undifferentiated spondyloarthropathy.Distinguishing features include sacroileitis with or withoutspondylitis; inflammatory asymmetric arthritis; association with HLA-B27(a serologically defined allele of the HLA-B locus of class I MHC);ocular inflammation, and absence of autoantibodies associated with otherrheumatoid disease. The cell most implicated as key to induction of thedisease is the CD8+T lymphocyte, a cell which targets antigen presentedby class I MHC molecules. CD8+T cells may react against the class I MHCallele HLA-B27 as if it were a foreign peptide expressed by MHC class Imolecules. It has been hypothesized that an epitope of HLA-B27 may mimica bacterial or other microbial antigenic epitope and thus induce a CD8+T cells response.

Systemic sclerosis (scleroderma) has an unknown etiology. A hallmark ofthe disease is induration of the skin which is likely induced by anactive inflammatory process. Scleroderma can be localized or systemic.Vascular lesions are common, and endothelial cell injury in themicrovasculature is an early and important event in the development ofsystemic sclerosis. An immunologic basis is implied by the presence ofmononuclear cell infiltrates in the cutaneous lesions and the presenceof anti-nuclear antibodies in many patients. ICAM-1 is often upregulatedon the cell surface of fibroblasts in skin lesions suggesting that Tcell interaction with these cells may have a role in the pathogenesis ofthe disease. Other organs may also be involved. In the gastrointestinaltract, smooth muscle atrophy and fibrosis can result in abnormalperistalsis/motility. In the kidney, concentric subendothelial intimalproliferation affecting small arcuate and interlobular arteries canresult in reduced renal cortical blood flow and thus proteinuria,azotemia and hypertension. In skeletal and cardiac muscle, atrophy,interstitial fibrosis/scarring, and necrosis can occur. Finally, thelung can have interstitial pneumonitis and interstitial fibrosis.

Idiopathic inflammatory myopathies including dermatomyositis,polymyositis and others are disorders of chronic muscle inflammation ofunknown etiology resulting in muscle weakness. Muscleinjury/inflammation is often symmetric and progressive. Autoantibodiesare associated with most forms. These myositis-specific autoantibodiesare directed against and inhibit the function of components involved inprotein synthesis.

Sjögren's syndrome is the result of immune-mediated inflammation andsubsequent functional destruction of the tear glands and salivaryglands. The disease can be associated with or accompanied byinflammatory connective tissue diseases. The disease is associated withautoantibody production against Ro and La antigens, both of which aresmall RNA-protein complexes. Lesions result in keratoconjunctivitissicca, xerostomia, with other manifestations or associations includingbilary cirrhosis, peripheral or sensory neuropathy, and palpablepurpura.

Systemic vasculitis are diseases in which the primary lesion isinflammation and subsequent damage to blood vessels which results inischemia/necrosis/degeneration to tissues supplied by the affectedvessels and eventual end-organ dysfunction in some cases. Vasculitidescan also occur as a secondary lesion or sequelae to otherimmune-inflammatory mediated diseases such as rheumatoid arthritis,systemic sclerosis, etc, particularly in diseases also associated withthe formation of immune complexes. Diseases in the primary systemicvasculitis group include: systemic necrotizing vasculitis: polyarteritisnodosa, allergic angiitis and granulomatosis, polyangiitis; Wegener'sgranulomatosis; lymphomatoid granulomatosis; and giant cell arteritis.Miscellaneous vasculitides include: mucocutaneous lymph node syndrome(MLNS or Kawasaki's disease), isolated CNS vasculitis, Behet's disease,thromboangiitis obliterans (Buerger's disease) and cutaneous necrotizingvenulitis. The pathogenic mechanism of most of the types of vasculitislisted is believed to be primarily due to the deposition ofimmunoglobulin complexes in the vessel wall and subsequent induction ofan inflammatory response either via ADCC, complement activation, orboth.

Sarcoidosis is a condition of unknown etiology which is characterized bythe presence of epithelioid granulomas in nearly any tissue in the body;involvement of the lung is most common. The pathogenesis involves thepersistence of activated macrophages and lymphoid cells at sites of thedisease with subsequent chronic sequelae resultant from the release oflocally and systemically active products released by these cell types.

Autoimmune hemolytic anemia including autoimmune hemolytic anemia,immune pancytopenia, and paroxysmal noctural hemoglobinuria is a resultof production of antibodies that react with antigens expressed on thesurface of red blood cells (and in some cases other blood cellsincluding platelets as well) and is a reflection of the removal of thoseantibody coated cells via complement mediated lysis and/orADCC/Fc-receptor-mediated mechanisms.

In autoimmune thrombocytopenia including thrombocytopenic purpura, andimmune-mediated thrombocytopenia in other clinical settings, plateletdestruction/removal occurs as a result of either antibody or complementattaching to platelets and subsequent removal by complement lysis, ADCCor FC-receptor mediated mechanisms.

Thyroiditis including Grave's disease, Hashimoto's thyroiditis, juvenilelymphocytic thyroiditis, and atrophic thyroiditis, are the result of anautoimmune response against thyroid antigens with production ofantibodies that react with proteins present in and often specific forthe thyroid gland. Experimental models exist including spontaneousmodels: rats (BUF and BB rats) and chickens (obese chicken strain);inducible models: immunization of animals with either thyroglobulin,thyroid microsomal antigen (thyroid peroxidase).

Diabetes mellitus is a genetic disorder of metabolism of carbohydrate,protein and fat associated with a relative or absolute insufficiency ofinsulin secretion and with various degrees of insulin resistance. In itsfully developed clinical expression, it is characterized by fastinghyperglycemia and in the majority of long-standing patients byatherosclerotic and microangiopathic vascular disease and neuropathy.Differences between various forms of the disease are expressed in termsof cause and pathogenesis, natural history, and response to treatment.Thus, diabetes is not a single disease but a syndrome.

Type I, or insulin-dependent diabetes mellitus (IDDM) occurs inapproximately 10 percent of all diabetic patients in the Western world.Type I diabetes mellitus or insulin-dependent diabetes is the autoimmunedestruction of pancreatic islet β-cells; this destruction is mediated byauto-antibodies and auto-reactive T cells. Antibodies to insulin or theinsulin receptor can also produce the phenotype ofinsulin-non-responsiveness.

Classically, this type of disease occurs most commonly in childhood andadolescence; however, it can be recognized and become symptomatic at anyage. In the most common type of IDDM (Type IA), it has been postulatedthat environmental (acquired) factors such as certain viral infections,and possibly chemical agents, superimposed on genetic factors, may leadto cell-mediated autoimmune destruction of β cells. Thus, geneticallydetermined abnormal immune responses (linked to HLA associations)characterized by cell mediated and humoral autoimmunity are thought toplay a pathogenetic role after evocation by an environmental factor. Asecond type of IDDM (Type IB) is believed to be due to primaryautoimmunity. These patients have associated autoimmune endocrinediseases such as Hashimoto's thyroiditis, Graves' disease, Addison'sdisease, primary gonadal failure, and associated nonendocrine autoimmunediseases such as pernicious anemia, connective tissue diseases, celiacdisease and myasthenia gravis. Insulin dependency implies thatadministration of insulin is essential to prevent spontaneous ketosis,coma, and death. However, even with insulin treatment, diabetic patientscan still have many of the additional problems associated with diabetes,i.e. connective tissue disorders, neuropathy, etc.

The second type of diabetes, Type II or non-insulin-dependent diabetesmellitus (NIDDM), present in approximately 90% of all diabetics, alsohas a genetic basis. Patients with type II diabetes may have a bodyweight that ranges from normal to excessive. Obesity and pathologicalinsulin resistance are by no means essential in the evolution of NIDDM.In the majority of patients with NIDDM, a diagnosis is made in middleage. Patients with NIDDM are non-insulin-dependent for prevention ofketosis, but they may require insulin for correction of symptomatic ornonsymptomatic persistent fasting hyperglycemia if this cannot byeachieved with the use of diet or oral agents. Thus, therapeuticadministration of insulin does not distinguish between IDDM and NIDDM.In some NIDDM families, the insulin secretory responses to glucose areso low that they may resemble those of early Type I diabetes at anypoint in time. Early in its natural history, the insulin secretorydefect and insulin resistance may be reversible by treatment (ie. weightreduction) with normalization of glucose tolerance. The typical chroniccomplications of diabetes, namely macroangiopathy, microangiopathy,neuropathy, and cataracts seen in IDDM are seen in NIDDM as well.

Other types of diabetes include entities secondary to or associated withcertain other conditions or syndromes. Diabetes may be secondary topancreatic disease or removal of pancreatic tissue; endocrine diseasessuch as acromegaly, Cushing's syndrome, pheochromocytoma, glucagonoma,somatostatinoma, or primary aldosteronism; the administration ofhormones, causing hyperglycemia; and the administration of certain drugs(i.e. antihypertensive drugs, thiazide diuretics, preparationscontaining estrogen, psychoactive drugs, sympathomimetic agents).Diabetes may be associated with a large number of genetic syndromes.Finally, diabetes may be associated with genetic defects of the insulinreceptor or due to antibodies to the insulin receptor with or withoutassociated immune disorders.

Immune mediated renal diseases, including glomerulonephritis andtubulointerstitial nephritis, are the result of antibody or T lymphocytemediated injury to renal tissue either directly as a result of theproduction of autoreactive antibodies or T cells against renal antigensor indirectly as a result of the deposition of antibodies and/or immunecomplexes in the kidney that are reactive against other, non-renalantigens. Thus other immune-mediated diseases that result in theformation of immune-complexes can also induce immune mediated renaldisease as an indirect sequelae. Both direct and indirect immunemechanisms result in inflammatory response that produces/induces lesiondevelopment in renal tissues with resultant organ function impairmentand in some cases progression to renal failure. Both humoral andcellular immune mechanisms can be involved in the pathogenesis oflesions.

Demyelinating diseases of the central and peripheral nervous systems,including multiple sclerosis; idiopathic demyelinating polyneuropathy orGuillain-Barre syndrome; and Chronic Inflammatory DemyelinatingPolyneuropathy, are believed to have an autoimmune basis and result innerve demyelination as a result of damage caused to oligodendrocytes orto myelin directly. In MS there is evidence to suggest that diseaseinduction and progression is dependent on T lymphocytes. Multiplesclerosis is a demyelinating disease that is T lymphocyte-dependent andhas either a relapsing-remitting course or a chronic progressive course.The etiology is unknown; however, viral infections, geneticpredisposition, environment, and autoimmunity all contribute. Lesionscontain infiltrates of predominantly T lymphocyte mediated, microglialcells and infiltrating macrophages; CD4+T lymphocytes are thepredominant cell type at lesions. The mechanism of oligodendrocyte celldeath and subsequent demyelination is not known but is likely Tlymphocyte driven.

Inflammatory and fibrotic lung disease, including eosinophilicpneumonias, idiopathic pulmonary fibrosis, and hypersensitivitypneumonitis may involve a disregulated immune-inflammatory response.Inhibition of that response would be of therapeutic benefit and withinthe scope of the invention.

Autoimmune or immune-mediated skin disease, including bullous skindiseases, erythema multiforme, and contact dermatitis are mediated byauto-antibodies, the genesis of which is T lymphocyte-dependent.

Psoriasis is a T lymphocyte-mediated inflammatory disease. Lesionscontain infiltrates of T lymphocytes, macrophages and antigen processingcells, and some neutrophils.

Transplantation associated diseases, including Graft rejection andGraft-Versus-Host-Disease (GVHD) are T lymphocyte-dependent; inhibitionof T lymphocyte function is ameliorative.

Other diseases in which intervention of the immune and/or inflammatoryresponse have benefit are infectious disease including but not limitedto viral infection (including but not limited to AIDS, hepatitis A, B,C, D, E and herpes) bacterial infection, fungal infections, andprotozoal and parasitic infections (molecules (or derivatives/agonists)which stimulate the MLR can be utilized therapeutically to enhance theimmune response to infectious agents), diseases of immunodeficiency(molecules/derivatives/agonists) which stimulate the MLR can be utilizedtherapeutically to enhance the immune response for conditions ofinherited, acquired, infectious induced (as in HIV infection), oriatrogenic (i.e. as from chemotherapy) immunodeficiency, and neoplasia.

Additionally, inhibition of molecules with proinflammatory propertiesmay have therapeutic benefit in reperfusion injury; stroke; myocardialinfarction; atherosclerosis; acute lung injury; hemorrhagic shock; burn;sepsis/septic shock; acute tubular necrosis; endometriosis; degenerativejoint disease and pancreatis.

The compounds of the present invention, e.g. polypeptides or antibodies,are administered to a mammal, preferably a human, in accord with knownmethods, such as intravenous administration as a bolus or by continuousinfusion over a period of time, by intramuscular, intraperitoneal,intracerebrospinal, subcutaneous, intra-articular, intrasynovial,intrathecal, oral, topical, or inhalation (intranasal, intrapulmonary)routes.

It may be desirable to also administer antibodies against other immunedisease associated or tumor associated antigens, such as antibodieswhich bind to CD20, CD11a, CD 40, CD18, ErbB2, EGFR, ErbB3, ErbB4, orvascular endothelial growth factor (VEGF). Alternatively, or inaddition, two or more antibodies binding the same or two or moredifferent antigens disclosed herein may be coadministered to thepatient. Sometimes, it may be beneficial to also administer one or morecytokines to the patient. In one embodiment, the polypeptides of theinvention are coadministered with a growth inhibitory agent. Forexample, the growth inhibitory agent may be administered first, followedby a polypeptide of the invention. However, simultaneous administrationor administration first is also contemplated. Suitable dosages for thegrowth inhibitory agent are those presently used and may be lowered dueto the combined action (synergy) of the growth inhibitory agent and thepolypeptide of the invention.

For the treatment or reduction in the severity of immune relateddisease, the appropriate dosage of an a compound of the invention willdepend on the type of disease to be treated, as defined above, theseverity and course of the disease, whether the agent is administeredfor preventive or therapeutic purposes, previous therapy, the patient'sclinical history and response to the compound, and the discretion of theattending physician. The compound is suitably administered to thepatient at one time or over a series of treatments.

For example, depending on the type and severity of the disease, about 1μg/kg to 15 mg/kg (e.g. 0.1-20 mg/kg) of polypeptide or antibody is aninitial candidate dosage for administration to the patient, whether, forexample, by one or more separate administrations, or by continuousinfusion. A typical daily dosage might range from about 1 μg/kg to 100mg/kg or more, depending on the factors mentioned above. For repeatedadministrations over several days or longer, depending on the condition,the treatment is sustained until a desired suppression of diseasesymptoms occurs. However, other dosage regimens may be useful. Theprogress of this therapy is easily monitored by conventional techniquesand assays.

G. Articles of Manufacture

In another embodiment of the invention, an article of manufacturecontaining materials useful for the diagnosis or treatment of thedisorders described above is provided. The article of manufacturecomprises a container and an instruction. Suitable containers include,for example, bottles, vials, syringes, and test tubes. The containersmay be formed from a variety of materials such as glass or plastic. Thecontainer holds a composition which is effective for diagnosing ortreating the cartilaginous disorder, and may have a sterile access port(for example the container may be an intravenous solution bag or a vialhaving a stopper pierceable by a hypodermic injection needle). Theactive agent in the composition will typically be an insulin or insulinvariant polypeptide. The composition can comprise any or multipleingredients disclosed herein. The instruction on, or associated with,the container indicates that the composition is used for diagnosing ortreating the condition of choice. For example, the instruction couldindicate that the composition is effective for the treatment ofosteoarthritis, rheumatoid arthritis or any other cartilaginousdisorder. The article of manufacture may further comprise a secondcontainer comprising a pharmaceutically-acceptable buffer, such asphosphate-buffered saline, Ringer's solution and dextrose solution.Alternatively, the composition may contain any of the carriers,excipients and/or stabilizers mentioned herein under section E.Pharmaceutical Compositions and Dosages. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, syringes, and package insertswith instructions for use.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

EXAMPLES

Commercially available reagents referred to in the examples were usedaccording to manufacturer's instructions unless otherwise indicated. Thesource of those cells identified in the following examples, andthroughout the specification, by ATCC accession numbers is the AmericanType Culture Collection, Manassas, Va. Unless otherwise noted, thepresent invention uses standard procedures of recombinant DNAtechnology, such as those described hereinabove and in the followingtextbooks: Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Press N.Y., 1989; Ausubel et al., Current Protocols inMolecular Biology, Green Publishing Associates and Wiley Interscience,N.Y., 1989; Innis et al., PCR Protocols: A Guide to Methods andApplications, Academic Press, Inc., N.Y., 1990; Harlow et al.,Antibodies: A Laboratory Manual, Cold Spring Harbor Press, Cold SpringHarbor, 1988; Gait, M. J., Oligonucleotide Synthesis, IRL Press, Oxford,1984; R. I. Freshney, Animal Cell Culture, 1987; Coligan et al., CurrentProtocols in Immunology, 1991.

Example 1 Effect on Insulin on Primary Articular Chondrocytes

Introduction

This experiment shows the effect of various concentrations (0.1-100 nM)of insulin on matrix (proteoglycan) synthesis and on viability ofchondrocytes in serum-free culture media. In order to culturechondrocytes, articular cartilage is digested with enzymes which removethe extracellular matrix. Thus, the cellular environment in this culturesystem may be similar to that found in later stages of cartilagedisorders where the matrix has been depleted. Since essentially all ofthe matrix synthesized by chondrocytes cultured in monolayer is secretedinto the media, the amount of proteoglycans in the media of such cellsis indicative of matrix synthesis. Proteoglycans are measured in mediausing the 1,9-dimethylmethylene blue (DMMB) colorimetric assay ofFarndale and Buttle, Biochim. Biophys. Acta 883: 173-177 (1986). In thisassay, the change in color of the DMMB dye which occurs upon its bindingto proteoglycans is quantitated spectrophotometrically. Chondrocyteviability was determined using a colorimetric assay that measures themetabolic activity of cultured cells based on the ability of viablecells to cleave the yellow tetrazolium salt MTT to form purple formazancrystals Berridge, M. V. & Tan, A. S., Arch. Biochem. Biophys. 303: 474(1993). The formazan crystals formed are solubilized andspectrophotometrically quantitated using an ELISA reader.

Materials and Methods

Chondrocyte Preparation:

The metacarpophalangeal joints of 4-6 month old female pigs wereaseptically dissected, and articular cartilage was removed by free-handslicing taking care so as to avoid the underlying bone. These cartilagefragments were then digested in 0.05% trypsin in serum-free Ham's F12for 25 minutes at 37° C. The medium was drained and discarded, andcartilage was digested in 0.3% collagenase B in serum-free Ham's F12media for thirty minutes at 37° C. The medium was drained and discarded,and the cartilage was digested overnight in 0.06% collagenase B in Ham'sF12+10% fetal bovine serum. The cells were then filtered through a 70micron nylon filter and seeded in Ham's F12 medium without serum.

Culturing of Chondrocytes:

Chondrocytes (prepared as described above) were grown in microtiterplates (Falcon microtest 96, flat bottom) at a density of 80,000 cellsper well in media composed of Ham's F12 with antibiotics (10 μg/mlgentamicin, 250 ng/ml amphotericin B, 100 μg/ml penicillin/streptomycin)in a final volume of 250 μl per well, for 6 days at 37° C. and 5% CO₂.Media was removed and used to measure proteoglycans at days 3 and 6.

Measurement of Proteoglycans:

DMMB is a dye that undergoes metachromasia (a change in color, in thiscase from blue to purple) upon binding to sulfated glycosaminoglycans(GAG), the side-chains of proteoglycans. The addition of sulfatedproteoglycans to DMMB causes a decrease in the peak values at 590 and660 nm with an increase in absorbance at 530 nm. Thus, the amount ofproteoglycans in media was determined by adding DMMB dye in a 96 wellplate format, and the change in color was quantitated using aspectrophotometer (Spectramax 250). The DMMB assay is a well-acceptedmethod to measure the amount of proteoglycans in cartilage cultures. Forthis assay, a standard curve was prepared using chondroitin sulfateranging from 0.0 to 5.0 μg.

MTT Assay:

After 6 days of treatment, 10 ul of the tetrazolium salt, MTT (stock 5mg/ml), (Boehringer Mannheim, cat. No.1465007) was added to theremaining 100 μl of media in each well. After another 4 hour incubationat 37° C. and 5% CO₂, 100 μl solubilization solution (reagent in assaykit from Boehringer Mannheim) was added and the plate was incubatedovernight at 37° C. and 5% CO₂. The absorbance was then measured at 584nm (and at 690 nm to determine background absorbance).

Results and Discussion

As show in FIG. 1, insulin increased synthesis of proteoglycans in adose-dependent manner. In addition, insulin (at concentrations as low as0.1 nM) inhibited the down-regulation of PG synthesis induced by IL-1α(right side of graph in FIG. 1). Thus, insulin (Intergen, Purchase, NewYork, cat. no. 450100) was a very potent stimulator of proteoglycansynthesis, and was able overcome the inhibitory effects of IL-1α (R&DSystems, cat. no. 200LA002) on proteoglycan synthesis.

Unlike most primary cells, chondrocytes are able to survive inserum-free media in the absence of any additional growth factors for atleast one week. However, addition of insulin (at 100 nM and 10 nM)increased the metabolic activity of chondrocytes cultured in this wayfor 6 days (FIG. 2). Insulin also blocked the ability of interleukin 1-αto inhibit the metabolic activity of chondrocytes (right half of graph).Such an activity could be very useful therapeutically, especially inconditions such as arthritis, joint trauma, or aging, where themetabolic activity of chondrocytes is compromised.

The ability of insulin to counteract the detrimental effects of IL1-αmakes insulin a very attractive candidate for the treatment ofconditions such as arthritis in which high levels of IL-1 are implicatedin disease progression.

Example 2 Articular Cartilage Explant Assay

Introduction

The experiments of this example examine both the synthetic andprophylactic potential of the test compound on cartilage matrixturnover. This potential is determined by measuring matrix (i.eproteoglycan) synthesis and breakdown, as well as nitric oxideproduction, in articular cartilage. These parameters are evaluated inthe presence and absence of interleukin 1α, IL-1α. Articular cartilageexplants have several advantages over primary cells in culture. First,and perhaps most importantly, cells in explants remain embedded intissue architecture produced in vivo. Secondly, these explants arephenotypically stable for several weeks ex vivo, during which time theyare able to maintain tissue homeostasis. Finally, unlike primary cells,explants can be used to measure matrix breakdown. To set up cartilageexplants, articular cartilage must be dissected and minced which resultsin disruption of the collagen network and release of proteoglycans intothe culture media. This system thus mimics degenerative conditions suchas arthritis in which the matrix is progressively depleted. Using thissystem, we have found that the test compound can: (1) stimulateproteoglycan (PG) synthesis; (2) inhibit PG release; (3) inhibitIL-1α-induced PG breakdown; (4) inhibit the IL-1α-induced reduction inPG synthesis; and (5) decrease both basal and IL-1α-induced nitric oxideproduction.

Il-1α has catabolic effects on cartilage including up-regulation ofenzymes which induce matrix breakdown (matrix metalloproteinases andaggrecanases) as well as inhibition of synthesis of new matrix molecules(proteoglycans and collagens). Thus, the ability of the test compound tonot only have positive effects on cartilage, but to also counteract thedeleterious effects of IL-1α is strong evidence of the protective effectexhibited by the test compound. In addition, such an activity suggeststhat the test compound could inhibit the degradation which occurs inarthritic conditions, since high levels of IL-1 are found in arthriticjoints, and since antagonism of IL-1 function has been shown to reducethe progression of osteoarthritis. Arend W. P. et al., Ann. Rev.Immunol. 16: 27-55 (1998).

The role of nitric oxide (NO) in the breakdown of articular cartilage,especially the destruction associated with osteoarthritis has beendescribed (Ashok et al., Curr. Opin. Rheum. 10: 263-268 (1998)). In vivoanimal models suggest that inhibition of nitric oxide production reducesprogression of arthritis (Pelletier, J P et al., Arthritis Rheum. 7:1275-86 (1998); van de Loo et al., Arthritis Rheum. 41: 634-46 (1998);Stichtenoth, DO and Frolich J. C., Br. J. Rheumatol. 37: 246-57 (1998).In humans, many of the drugs used to treat rheumatoid arthritis candecrease nitric oxide production or activity. Since NO also has effectson cell types besides chondrocytes, the presence of NO within the jointmay also increase vasodilation and permeability, potentiate cytokinerelease by leukocytes, and stimulate angiogenic activity. Becauseproduction of NO by cartilage correlates with disease state, and sinceNO appears to play a role in both erosive and inflammatory components ofjoint diseases, a factor which decreases nitric oxide production wouldlikely be beneficial in the treatment of cartilaginous disorders.

The assay for nitric oxide described herein is based on the principlethat 2,3-diaminonapthalene (DAN) reacts with nitrite under acidicconditions to form 1-(H)-naphthotriazole, a fluorescent product. As NOis quickly metabolized into nitrite (NO₂ ⁻¹) and nitrate (NO₃ ⁻¹),detection of nitrite is one means of detecting (albeit undercounting)the actual NO produced in cartilaginous tissue.

Materials and Methods

Articular Cartilage Explants:

The metacarpophalangeal joint of 4-6 month old female pigs wasaseptically dissected as described above. The cartilage was minced,washed and cultured in bulk for at least 24 hours at 37° C. and 5% CO₂in explant media, i.e. serum free (SF) LG DMEM/F12 media with 0.1% BSA,100 U/ml penicillin/streptomycin (Gibco), 2 mM L-Glutamine, 0.1 mMsodium pyruvate (Gibco), 20 μg/ml Gentamicin (Gibco) and 1.25 mg/LAmphotericin B. Articular cartilage was aliquoted into micronics tubes(approximately 55 mg per tube) and incubated for at least 24 hours inthe above media. Media was harvested and new media was added (alone orwith fresh insulin) at various time points (0, 24, 48 and 72 hours).

Proteoglycan Release:

Media was harvested at various time points was assayed for proteoglycancontent using the 1,9-dimethylmethylene blue (DMMB) colorimetric assayof Farndale and Buttle, Biochim. Biophys. Acta 883: 173-177 (1985) asdescribed above. PG release at 0 hours was used as a baselinemeasurement, and any samples with especially high or low PG release werediscarded prior to treatment with insulin. For all treatments, resultsrepresent the average of 5 independent samples.

Proteoglycan Synthesis:

At 48 hours after the first treatment, ³⁵S-sulfate was added tocartilage explants at a final concentration of 10 μCi/ml along withfresh media (with or without test compound). After an additional 12-17hours of incubation at 37° C., the media was removed and saved forsubsequent PG and nitric oxide (NO) analysis. The cartilage explantswere washed twice with explant media and digested overnight at 50° C. ina 900 mL reaction volume of 10 mM EDTA, 0.1M sodium phosphate and 1mg/ml proteinase K (Gibco BRL). The digestion reaction was mixed (2:1)with 10% w/v cetylpyridinium chloride (Sigma) to precipitate theproteoglycans and centrifuged at 1000×g for 15 minutes. The supernatantwas removed and formic acid (500 ml, Sigma) was added to dissolve thepellets. The samples were then transferred to vials containing 10 mlscintillation fluid (ICN) and read in a scintillation counter.

Remaining Proteoglycan in Cartilage Tissues:

After 72 hours, the remaining articular cartilage explants were digestedas described above under Proteoglycan synthesis and assayed forproteoglycan content using the DMMB calorimetric assay (referenced aboveunder Proteoglycan release).

Nitric Oxide Assay:

Articular cartilage media saved from the cartilage explants at varioustimes (24, 48, and 72 hours) was mixed with 10 μl 0.05 mg/ml2,3-diaminonapthalene (DAN) in 0.62M HCl and incubated at roomtemperature for 10-20 minutes in the dark. The reaction was terminatedwith 5 μl of 2.8N NaOH. The amount of fluorescence of2,3-diamionaphthotriazole was measured with a Cytoflor fluorescent platereader at 365 nm excitation at 409 nm emission.

Results and Discussion

Similar to its effects on primary articular chondrocytes (FIG. 1),insulin (Intergen, Purchase, New York, cat. no. #450100) inducedproteoglycan synthesis in articular cartilage explants and could atleast partially block the inhibitory effects of IL-1α (R&D Systems, cat.no. 200LA002)(FIG. 3B). In addition, insulin decreased the breakdown ofmatrix which occurs in absence or presence of IL-1α (FIG. 3A). Sincematrix breakdown is one of the earliest and most destructive features ofarthritis, inhibition of this process and stimulation of new matrixmolecules should promote tissue and joint repair. Most importantly, thisdecrease in matrix breakdown and increase in synthesis induced byinsulin resulted in an increase in the total amount of proteoglycans inthe articular cartilage explants relative to that of untreated tissues(FIG. 4). By increasing the amount of matrix retained in cartilage,insulin treatment in vivo would lead to retention of articular cartilagematrix, and thus inhibition of subsequent joint destruction anddeformity.

In addition to its ability to decrease matrix breakdown and increasematrix synthesis, insulin also inhibited nitric oxide (NO) production byeither untreated or IL-1α-treated articular cartilage explants (FIG. 5).This effect was seen at concentrations as low as 1 nM and was also seenwith IGF-treated explants. As described above, nitric oxide hasdetrimental effects on chondrocytes as well as other cell types withinthe joint. Since inhibition of nitric oxide has been shown to inhibitprogression of arthritis in animals, the effect of insulin on NO furthersuggests that insulin would be protective for joint tissues in vivo.

Example 3 Mouse Patellae Assay

Introduction

This assay determines the in vitro and in vivo effect of the testedcompound on proteoglycan synthesis in the patellae of mice. The patellais a very useful model to study the effects of the test compound becauseit permits the evaluation on cartilage which has not been removed fromthe underlying bone. Moreover, since each animal has one patellae ineach leg, experiments can be performed using the contralateral joint asa control. This assay involves injection of a protein into theintra-articular space of a (mouse) knee joint, and subsequent harvest(within a few days after injection) of the patella (kneecap) formeasurement of matrix synthesis (FIG. 6). The procedure performedherein, and outlined in FIG. 6, has been previously used to measureeffects of cytokines in vitro and in vivo (Van den Berg et al., Rheum.Int. 1: 165-9 (1982); Vershure P. J. et al., Ann. Rheum. Dis. 53:455-460 (1994); and Van de Loo et al., Arthit. Rheum. 38: 164-172(1995)).

Materials and Methods

In Vitro Treatment of Patellae

Patellae of 2 month old C57B1-6J mice (Jackson Laboratories) weredissected away from surrounding soft tissue and incubated overnight inexplant media (see above, example 2) with no additional factors or with100 ng/ml IL1α or 100 nM insulin. Patellae were labelled with ³⁵S-sulfur(30 μCi/ml) during the last 3 hours of the 18 hour incubation, and thenwashed three times with phosphate buffered saline (PBS). Samples werefixed overnight in 10% formalin followed by decalcification of theunderlying bone in 5% formic acid. Cartilage was dissected away from theunderlying bone, placed in 500 μl of a tissue and gel solubilizer(Solvable, Packard Instrument Company) and incubated at 60° C. for 1.5hours. Scintillation fluid designed for concentrated alkaline and saltsolutions (HIONIC-fluor, Packard Instrument Company) was added (10 mL)to each tube and mixed thoroughly. ³⁵S uptake was then measured using ascintillation counter. The levels of PG synthesis are reported as cpmand show the average of patellae from 4 different mice/group.

In Vivo Treatment of Patellae

Mice were separated into two subgroups and injected and either a highdose (6 μl) or a low dose (3 μl) of the test compound (e.g., insulin @10mg/ml) was injected intra-articularly into the right knee daily forthree consecutive days. The patellae were then harvested, labelled for 3hours in explant media without any additional factors, and processed asdescribed above. FIG. 7B displays the data as a ratio of theincorporation into the treated versus untreated knee. Datapointsrepresenting a treated/untreated ratio of greater than 1 (above theline) indicate that treatment of the tested compound resulted inincreased proteoglycan synthesis. PG synthesis as a result ofapplication of the high dose (6 μl) is indicated by the left datapoints,while the low dose (3 μl) is indicated by the right datapoints. Eachpoint represents results from an individual mouse.

Results and Discussion

Similar to its induction of proteoglycan (PG) synthesis in vitro in thedissected cartilage explants (example 2), insulin stimulated PGsynthesis in intact cartilage both in vitro (FIG. 7A) and in vivo (FIG.7B). More specifically, injection of insulin (Intergen, Purchase, NewYork, cat. no. 450100) into normal mouse knee joints resulted in anapproximately 25% increase in PG synthesis in 3 days (FIG. 7B). Such anincrease would likely have a beneficial effect on diseased tissues wherecartilage matrix, lost through degradation, can not be replaced.Furthermore, this percentage increase may under-estimate the beneficialeffects of insulin on cartilage matrix in vivo, since the ability ofinsulin to decrease cartilage breakdown (see example 2) would not havebeen detected by this assay and thus not be included in thiscalculation. The decrease in breakdown induced by insulin would furtherincrease the amount of matrix retained in insulin-treated knee joints.Finally, these experiments represent initial attempts to determinesafety in an animal model. Rather surprisingly, no adverse effects wereseen upon intra-articular injection of very high doses (30 μg) ofinsulin once/day for 3 days.

Thus, the test compound insulin had positive effects on cartilage withinin the joint space, and intra-articular injection of purified insulinprotein into knee joints appears to be a tenable treatment strategy.

Example 4 Guinea Pig Model

Introduction

These experiments measure the effect of the test compound onproteoglycan (PG) synthesis and breakdown in the cartilage of DunkinHartley (DH) Guinea Pigs, an accepted animal model for osteoarthritis(Young et al., “Osteoarthrits”, Spontaneous animal models of humandisease vol. 2, pp. 257-261, Acad. Press, New York. (1979); Bendele etal., Arthritis Rheum. 34: 1180-1184; Bendele et al., Arthritis Rheum.31: 561-565 (1988); Jimenez et al., Laboratory Animal Sciences vol47(6): 598-601 (1997). Unlike most other animal models which haverapidly progressing tissue breakdown in their joints, DH guinea pigshave naturally occurring, slowly progressive, non-inflammatory OA-likechanges in their joints.

The highly reproducible pattern of cartilage breakdown in these guineapigs is similar to that seen in the human disorder (FIG. 8). Whilejoints appear normal in 2 month old DH guinea pigs, as the animals age,the course and severity of the disease progresses in a manner similar tothat of human OA. Thus, early histologic change include focalchondrocyte degeneration and death and loss of matrix in the superficiallayer of articular cartilage. While surviving chondrocytes adjacent tothe hypocellular fibrillated area may synthesize proteoglycans, thesecells can not maintain normal matrix content and structure. Thesechondrocytes do not migrate into and distribute themselves normallywithin the hypocellular area—instead, they stay in clusters surroundedby proteoglycan. As a result, this cartilage matrix devoid of normallydistributed chondrocytes undergoes further degeneration, ultimatelyresulting in destruction of the articular surface (Bendele and Hulman,Arthritis Rheum. 31: 561-565 (1988)). Thus, over time, chondrocyte andmatrix loss become more extensive. A transient increase in chondrocyteproliferation leads to the formation of peripheral chondrophytes, whichsubsequently undergo endochondral ossification to form osteophytes.Thereafter, moderate to severe cartilage degeneration occurs, withextensive deep cartilage degeneration, osteophyte formation, subchondralbone thickening and synovial hyperplasia and fibrosis. Thus, the jointsof DH guinea pigs over 1 year old are severely affected with marginalosteophytes of the tibia and femur, sclerosis of the subchondral bone ofthe tibial plateau, femoral condyle cysts, and calcification of thecollateral ligaments. (Jimenez et al., supra).

Materials and Methods

Male Dunkin Hartley guinea pigs, obtained from Charles RiverLaboratories (Wilmington, Mass.) were separated into treatment groupsfor sacrifice at 1-2, 6 and 11 months of age. At sacrifice, themetacarpophalangeal joints were aseptically dissected, and articularcartilage was removed by free-hand slicing taking care so as to avoidthe underlying bone. Cartilage was minced, washed and cultured in bulkfor at least 24 hours in a humidified atmosphere of 37° C. and 5% CO₂ inserum free (SF) LG DMEMIF12 media with 0.1% BSA, 100 U/mlpenicillin/streptomycin (Gibco), 2 mM L-Glutamine, 0.1 mM sodiumpyruvate (Gibco), 20 μg/ml Genamicin (Gibco) and 1.25 mg/L AmphotericinB. Articular cartilage was aliquoted into Micronics tubes (approximately35 mg per tube) and incubated for at least 24 hours in the above media.Media was harvested and fresh media was added alone or with insulin atvarious time points (0, 24, 48 and 72 hours).

Proteoglycan Release:

Media harvested at various time points was assayed for proteoglycancontent using the 1,9-dimethylmethylene blue (DMMB) colorimetric assayof Farndale and Buttle, Biochem. Bhiophys. Acta 883: 173-177 (1985) asdescribed above (example 2, articular cartilage explant assay). PGrelease at 0 hours was used as a baseline measurement, and any sampleswith especially high or low PG release were discarded prior totreatment.

Proteoglycan Synthesis:

After the media change at 48 hours, ³⁵S-sulfate (final concentration of10 μCi/ml) was added to the cartilage explant cultures, and tissues wereprocessed as above under Example 2, Articular Cartilage Explant Assay).

Remaining Proteoglycan in Cartilage Tissues:

After 72 hours, the remaining articular cartilage explants were digestedas described above under Proteoglycan synthesis and assayed forproteoglycan content using the DMMB colorimetric assay (referenced aboveunder Proteoglycan release).

Results and Discussion

One factor which is known to affect cartilage matrix metabolism is age.Not only does the rate of biosynthesis and the ability to repair tissuedecrease with age, but responsiveness to a number of growth factors,including insulin-like growth factor (IGF-1), is compromised as well(Schafer et al. Arch. Biochem and Biophys. 302:431-438 (1993). Diseaseis also associated with a decrease in growth factor sensitivity, asevidenced by the finding that the response of arthritic cartilage toIGF-1 is significantly blunted relative to age-matched controls(Chevalier and Tyler 1996, Br. J. Rheum 35: 515-522; J. Posever et al.,J. Orthopaedic Res. 13: 832-827 (1995)). For this reason, we tested theeffect of insulin on tissue from guinea pigs at various ages, and thusvarious stages of degeneration. As shown in FIG. 9, insulin increasedproteoglycan (PG) synthesis to approximately the same extent (two-fold)in articular cartilage from 1-2, 6, or 11 month old DH guinea pigs.Thus, DH guinea pig cartilage responded to insulin at various ages andstages of disease. Besides increasing PG synthesis, insulin alsodecreased matrix breakdown, as shown by a decrease in the amount of PGsin the media of insulin-treated explants (FIG. 10, left side, “Media”).Most importantly, this insulin-induced decrease in breakdown andincrease in synthesis resulted in a significant net gain in the amountof proteoglycans remaining in insulin-treated cartilage, even in tissuefrom 11 month old animals treated for only 3 days (FIG. 10, right side“Tissue”). This data, which shows an increase in the amount of PGsmaintained in the matrix of insulin treated cartilage, strongly suggeststhat insulin would be a very effective and potent stimulator ofcartilage repair, since loss of matrix occurs early and continuouslythrough-out the disease process.

Example 5 Diabetic Mouse Model

Metabolic changes in patients with diabetes mellitus (DM) affect manyorgan systems. For example, patients with diabetes have an increasednumber of musculoskeletal injuries and disorders relative to patientswithout diabetes. In fact, diabetes is one of the known risk factors fordeveloping arthritis. Changes in proteoglycans have been found in theintervertebral disc of diabetic patients, and articular cartilagesamples from patients with diabetes mellitus have compromised structuralintegrity (Robinson et al. Spine 23:849-55 (1998); Athanasiou et al.,Clin Orthop 368, 182-9 (1999). The mechanism underlying these changes inarticular cartilage in diabetic patients is not yet known.

Syndromes resembling human diabetes occur naturally in many animals orcan be induced surgically by removal of pancreata or by treatment withdrugs, viruses, or a specific diet. Among these models, diabetes inducedby streptozotocin (STZ) administration is accompanied by defects ininsulin secretion and action which in many ways resemble those found inhuman non-insulin dependent diabetes (Portha, B. et al. Diabetes Metab.15:61-75, 1989). Streptozotocin (STZ)-induced diabetes in animal modelsis associated with atrophy and depressed collagen content of connectivetissues including skin, bone, and cartilage (Craig, R. G. et al.Biochim. Biophys. Acta 1402: 250-260.1998). Importantly, cartilage fromSTZ-induced rats was found to be resistant to the anabolic action ofIGF-I, as measured by ³⁵SO₄ incorporation in vitro (Kelley, K. M. et al.1993. Diabetes 42: 463-469). In order to better understand the effectsof diabetes on cartilage matrix metabolism and to test the effects ofinsulin in another model of diseased, potentially growth-factorresistant cartilage, we measured matrix synthesis in articular cartilagefrom STZ-induced diabetic mice cultured alone or in the presence ofinsulin.

Materials and Methods

Induction of Diabetes:

8 week old female CD-1 mice were injected with 40 mg/kg STZ for 5consecutive days. Patellae were harvested at 2, 3, or 5 months aftertreatment. Blood was also collected from the tail at these sametimepoints, and blood glucose levels were measured using a glucose meter(One-Touch, Lifescan).

Patella Assay:

Patellae were incubated in the absence of presence of insulin (100 nM,Intergen, Purchase, New York, cat. no. 450100) and ³⁵S-sulfate (finalconcentration 15 μCi/ml) for 18 hours. Patellae were dissected asprocessed as above (example 3).

Results and Discussion

At all timepoints tested (2, 3 and 5 months), articular cartilage fromSTZ-treated mice had lower basal levels of proteoglycan synthesis (FIG.11). This decrease in synthesis could be due to the fact thatSTZ-treatment results in low serum insulin levels due to destruction ofpancreatic cells which produce insulin (Portha, B et al. Diabetes Metab.15:61-75, 1989; Craig, R. G. et al. Biochim. Biophys. Acta 1402:250-260.1998; Kelley, K. M. et al. Diabetes 42: 463-469 (1993). Mostimportantly, insulin induced matrix synthesis in cartilage fromSTZ-treated mice to the same extent as in cartilage from normal mice. Infact, insulin was able to restore matrix synthesis in cartilage ofSTZ-treated mice to levels comparable to that of untreated, normal mice.In contrast, IGF-1 was not able to increase proteoglycan synthesis incartilage from STZ-treated animals (Kelley, K. M. et al. Diabetes 42:463-469 (1993). Therefore, diseased tissue is not equally responsive toanabolic factors, and insulin may prove to be more effective than IGF-1in diabetic animals.

Our data supports the hypothesis that insulin-deficiency in STZ-treatedmice leads to decreased matrix synthesis in cartilage. Furthermore,regardless of the mechanism whereby synthesis is lower in STZ-treatedmice, insulin treatment was able to restore synthesis to normal levels.These results suggest that insulin would be an effective treatment fordisorders (other than primary arthritis) in which cartilage tissue hasdefects in matrix synthesis and/or breakdown.

Example 6 Preparation and Analysis of Polymeric Microspheres ContainingRecombinant Human Insulin

Introduction

While intra-articular injections are generally well-tolerated bypatients and once/week injections of therapeutics are currently beingtested clinically, an ideal drug would be one in which a limited numberof doses was required. Unfortunately, human insulin (HI) is unstablewhen stored in neutral solutions at low concentration for extendedperiods of time. J. Brenge and L. Langkjoer, Insulin Formulation andDelivery in Protein Delivery Eds. L. M. Sanders and W. Hendren.Furthermore, insulin has a half-life of about 5 minutes in the humanbody. Hadley, M. E. Endocrinology, Prentice-Hall, Inc. 1988. Thus, astabilized, slow-release formulation of HI is highly desirable. Humaninsulin (Eli Lilly, Indianapolis, Ind.) has also been formulated withzinc acetate to produce a sparingly soluble Zn:H1 complex. The Zn:H1complex is believed to result in a longer-acting formulation of insulin.In fact, histochemical evidence indicates that HI is stored in thepancreas as a zinc complex. J. Brenge and L. Langkjoer, InsulinFormulation and Delivery in Protein Delivery Eds. L. M. Sanders and W.Hendren, Plenum Press, 1997; Hadley, M. E. Endocrinology, Prentice-Hall,Inc. 1988. In addition, HI complexed with zinc is known to be moreresistant to aggregation than uncomplexed HI. J. Brenge and L.Langkjoer, supra. Finally, insulin complexed with zinc has been shown tohave a slower onset and a longer duration of activity (up to 24 hours)in humans relative to regular insulin. J. Brenge and L. Langkjoer,supra.

The microsphere formulations were analyzed to determine size, proteinload, physical and biological integrity. The amount of human insulin(HI) encapsulated (w/w) was determined by chemical analysis as perCleland, J. C. and Jones, A. J. S., Pharm. Res. 13(10): 1464-1475(1996)]. The physical and biological integrity of the HI recovered fromthe microspheres was analyzed by size exclusion (SEC) and reverse-phasechromatography (RPC). SEC and RPC were used to detect aggregated anddeamidated HI, respectively.

Material and Methods

Microsphere Formulation:

In the microsphere formulations prepared, both the Zn:HI hexamer ratio(e.g., formulation I-2:1 and formulation II-4:1) while the proteinloading remained constant (approximately 5.0%). The molar ratio oflactide to glycolide in all polymers was kept constant at 50:50. Allmicrosphere formulations were prepared using D,L-PLGA obtained fromBoehringer Ingelheim (Ingelheim, Germany; RG502H; 0.2 dL/g, 8 kD).Recombinant human insulin (HI) was encapsulated into PLGA microspheresusing a cryogenic, non-aqueous process described by Gombotz et al., U.S.Pat. No. 5,019,400, issued May 28, 1991 and Johnson et. al. Nature Med 2(7):795-799 (1996).

In preparation for insertion into the microspheres, the above HIformulations were first spray-freeze dried. This process wasaccomplished by atomizing the above formulations through an ultrasonicnozzle (Sono-Tek, Milton N.Y.) into liquid nitrogen, followed bylyophilization as previously described (Johnson et al. supra). Forexample, the dried Zn—HI powder (100 mg) was added to 5.8 ml of a 0.17mg/ml solution of the D,L-PLGA described above in ethylacetate solventand homogenized for 2 minutes at 8000 rpm with a shear homogenizer(Vitrius Inc. Giandiner, N.Y.) in order to form a uniform suspension ofZn—HI and polymer. The polymer and Zn—HI suspension was sprayed througha sonicating nozzle (Sono-Tek, Milton N.Y.) into a vessel containing 300ml of frozen ethanol. The vessel was then placed in a −70° C. freezer(to raise the temperature to −70° C.) whereupon the frozen ethanolmelted and the micropheres slowly hardened as the ethylacetate solventwas extracted by the ethanol. After 3 days, the hardened microsphereswere harvested by filtration through a 20 μm screen then dried undernitrogen gas for 4 days and finally sieved through a 60 μm screen.

Chromatography:

Size exclusion (SEC) and reverse-phase chromatography (RPC) procedureswere performed as per published methods (Pietta, P. G. et al., J.Chromatogr. 549:367:373 (1991); Klyushnichenko V. E. et al., J.Chromatogr. 661:83-92 (1994). Briefly, the SEC was performed on aZorbax® Column with phosphate buffer as the mobile phase. HI wasdetected by UV absorption at 214 nm. Reverse-phased chromatography wascarried out on a C-18 reversed-phase column using sulfate buffer withacetonitrile as the mobile phase at 40° C., HI was detected by UVabsorption at 214 nm as previously described (Pietta, P. G. et al., J.Chromatogr. 549:367-373 (1991); Klyushnichenko, V. E. et al., J.Chromatogr. 661:83-92 (1994).

Protein Analysis:

Encapsulated HI was recovered form the microspheres by dissolving themin 1.0 N sodium hydroxide (NaOH) and the soluble protein was recoveredand analyzed by UV absorbances using the experimentally determinedabsorbance A (1 mg/mL, 291 nm, 1 cm)=1.94.

Results and Discussion

The mean particle diameter distribution of the microspheres was measuredon a Malvern Masterisizer X and were found to be about 30 microns (TableI). Protein loading of formulation I and formulation II was found to be5.56% and 5.59% respectively (Table I). The analysis of HI integrityindicated that there were no significant differences between the proteinbefore and after encapsulation as determined by an insulin receptorkinase assay KIRA (see Example 7 below for procedure).

TABLE I Physical properties of PLGA insulin microsphere formulationsFormulation Loading (% w/w) Diameter (nm) Insulin Zn Ratio^(a)Formulation I 5.56 30.2 ± 12 1:2 Formulation II 5.59 36.6 ± 11 1:4^(a)Ratio defined as moles insulin hexamer/moles of zinc

Example 7 Release of Insulin From HI Microspheres

Introduction

In order to determine the amount of insulin released, insulin-loadedmicrospheres were incubated under 3 different conditions, and therecovered protein was analyzed for activity at several timepoints.Initially, microspheres were incubated in 10 mM histidine buffer at pH7.4 at 37° C. In order to measure release under conditions similar tothat of the joint, microspheres were also incubated in either synovialfluid or with articular cartilage explants. In all cases, samples weretaken at several timepoints and analyzed by measuring induction ofinsulin receptor phosphorylation in cells expressing the human insulinreceptor.

Materials and Methods

Release of Insulin in Buffer:

To evaluate the in vitro release profile for HI microsphereformulations, 20 mg of each HI microsphere formulation was placed in 500μL of release buffer (10 mM Histidine, 10 mM NaCl, 0.02% Polysorbate 20,0.02% NaN3, pH 7.2) and incubated at 37° C. The entire release mediumwas replaced at each sampling interval and the resulting release samplesstored at 5° C. prior to analysis.

Release of Insulin in Synovial Fluid:

Synovial fluid was harvested from 7-8 week old male Sprague-Dawley ratsand diluted 1:2 with phosphate buffered saline (PBS). The entire releasemedium was replaced at each sampling interval and the resulting samplesstored at 5° C. prior to analysis.

Release of Insulin in Explant Cultures:

Media from articular cartilage explants, incubated with insulin (10 nM)or PLGA-Insulin microspheres was harvested at various timepoints andtested for activity in the insulin kinase receptor assay. PLGA-Ins beadswere resuspended in 500 ul of explant media, and 3 ul of this mixturewas added to each explant sample in 260 ul of explant media.

Insulin Kinase Receptor Assay (KIRA):

Cell stimulation: Chinese Hamster Ovary (CHO) cells transfected with thehuman insulin receptor were plated (100 μl of 5×10⁵ cells/mL) in flatbottom-96-well sterile tissue culture plates (Falcon 1270) in media(PS/20 with 5% diafiltered FBS and antibiotics 1×glutamine, 1×penn/strep, 10 μg/mL puromycin) and incubated overnight in humidifiedatmosphere of 37° C., 95% air and 5% CO₂. Cells were then treated with100 μl of sample in media (PS/20 with 0.5% BSA) and incubated for 15minutes at 37° C. 130 μl of Lysis Buffer (150 mM NaCl with 50 mM HEPESand 0.5% Triton-X 100) with protease (AEBSF, 1 mM, Aprotinin, Leupeptin0.05 mM) and phosphatase (Sodium orthovanidate, 20 mM) inhibitors wasadded to each well. Samples were then used for ELISA analysis.

Enzyme-linked immuno-specific assay (ELISA): Plates (Nunc Maxisorpimmunoplate 4-39454) were coated with 100 μl of 1° monoclonal antibody(final concentration 2 μg/ml in PBS pH 7.0) at 4° C. overnight. Afterremoval of this solution, plates were incubated with 150 μl of blockingbuffer (PBS with 0.5% BSA) for 1 hour. Plates were then washed threetimes with wash buffer (PBS with 0.05% tween 20 pH 7.4) (Skatron ScanWasher 300). 80 μL of lysis buffer (150 mM NaCl with 50 mM HEPES and0.5% Triton-X 100) and 20 μl sample (from above) were added to theprepared ELISA plates. Plates were incubated at room temperature for 2hrs with gentle agitation. After washing wells six times with washbuffer, samples were incubed with 100 μl of biotinylated 2° antibody(4G10, final concentration 0.1 μg/ml in assay buffer, PBS with 0.5% BSA,0.05% tween 20 and 5 mM EDTA, pH 7.4) (UBI) for 2 hours with gentleagitation. Plates were then washed six times in wash buffer, and sampleswere incubed with 100 μl of Streptavidin/HRP (diluted 1:50,000 in assaybuffer) (Amdex) at room temp. for 1 hour with gentle agitation. Plateswere then washed with wash buffer six times and incubated with 100 μl ofsubstrate solution (1 volume of K&P TMB substrate+1 volume of K&P TMBperoxide solution in TMB, substrate kit from Kirkegard and Perry). Aftercolor development (10-15 minutes), the reaction was quenched with 100 μlof 1.0 N H3PO₄. The O.D. at 450 nm was then read.

Results and Discussion

The insulin kinase receptor assay (KIRA) is a very sensitive assay whichmeasures active insulin. An initial burst of release (at day 1) ofprotein was seen in both buffer (FIG. 12) and in synovial fluid (FIG.13A). The release profiles of HI from formulation II (PLGA-Zn) indicateda biphasic release where almost 40% of the total loaded protein wasreleased in the first 24 hours, followed by a lag phase (<1% dailyrelease) and a second release phase (2-5% daily release) over the next15 days at which point 89% of the total protein loaded had been released(FIG. 12). After incubation in synovial fluid for 3 days, microspheresfrom formulation II continued to release active insulin with aconcentration of approximately 5 μM (1.67 mg/ml total). Thus, theseresults indicated that active insulin was released from the loadedmicrospheres and that the formulation process did not seem to bedetrimental to protein quality.

The advantage such a slow-release system might have in vivo isillustrated by experiments testing insulin and insulin-loadedmicrospheres after incubation with articular cartilage explants. In thissystem, samples are treated only once, media is harvested on subsequentdays, and cells are metabolically active during the course of theexperiment. In this way, the stability of insulin in a biologicallyrelevant system could be determined. While insulin remained fairlystable when cultured in media without tissue (at 37° C.) (FIG. 14A), inthe presence of articular cartilage, the amount of active insulindecreased dramatically. In fact, within 24 hours after incubation withcartilage, the amount of active insulin had decreased by as much as 70%,and by 4 days of culture, little active insulin was detected (FIG. 14B).In contrast, significant levels of active insulin were found as late as3 days after treatment with PLGA-Ins (14C). Furthermore, even whendiluted PLGA-Ins microspheres were incubated with tissue, the amount ofactive insulin in the PLGA-Ins samples at 3 days was almost 14 timeshigher than that in the insulin samples at 3 days (FIG. 14D).

In conclusion, we have shown that PLGA-Ins microspheres continue torelease active insulin over time in the presence of articular cartilage.Thus, insulin remains active and apparently unaltered after release frommicrospheres incubated in a biologically relevant system for severaldays. Taken together, our results suggest that PLGA-Ins which would beuseful for local, slow-release delivery of insulin to the joint.

Example 8 Biological Activity of PLGA-Ins

Introduction

To verify that the insulin released from the PLGA-Ins microspheres wasactive on articular cartilage, we added these microspheres to articularcartilage explants and harvested and changed the media (without addingmore PLGA-Ins) every day for three days. Activity was determined bymeasuring proteoglycan breakdown and synthesis as described above forarticular cartilage explants (Example 2). As a control, we treated someexplant samples with fresh insulin (10 nM) at each media change (i.e. at0, 24, and 48 hr timepoints).

Data from explants cultures can be used to predict the effect ofspecific proteins on articular cartilage in vivo. However, within thejoint several types of tissue besides cartilage are present. Inaddition, proteins can be rapidly cleared from the synovial fluid.Therefore, to determine whether or not insulin released from PLGA-Insmicrospheres had an effect on cartilage in vivo, we injected mouse kneejoints with PLGA-Ins microspheres and measured proteoglycan synthesis.

Materials and Methods

Effect of PLGA-Ins Microspheres on Proteoglycan Release and Synthesis:

Explants were treated with PLGA-Ins microspheres (3 μl of a solution ofmicrospheres resuspended in 0.5 ml of explant media were added toexplants in 260 ul of media) on day 0, and media was harvested andreplaced (without microspheres) on subsequent days. Proteoglycanbreakdown and synthesis were measured as described above (example 2,articular cartilage explants). Some explant samples were treated withfresh insulin (10 nM) at each media change (i.e. at 0, 24, and 48 hrtimepoints).

Intra-Articular Injections:

PLGA-Ins microspheres were resuspended in 500 μl of buffer (0.1%hyaluronic acid in phosphate-buffered saline), and 3 μl of this solutionwas injected intra-articularly into mousejoints. As a control, 3 μl ofbuffer was injected into the contralateral knee. Three days later,patellae were harvested and proteoglycan synthesis was determined asabove (example 3, patella assay).

Results and Discussion

Explants treated with PLGA-Ins microspheres showed decreased matrixbreakdown (FIG. 15A) and increased matrix synthesis (FIG. 15C). Inaddition, the ability of IL-1α to induce breakdown down (FIG. 15B) andinhibit matrix synthesis (FIG. 15D) were prevented by co-treatment withPLGA-Ins (FIGS. 15A-B). Finally, PLGA-Ins inhibited both basal (FIG.15E) and IL-1α induced (FIG. 15F) nitric oxide production. These resultssuggest that PLGA-Ins could inhibit the cartilage catabolism and losswhich occurs in cartilage disorders such as arthritis.

Three days after injection of PLGA-Ins into mouse knee joints, matrixsynthesis was significantly (p<0.05) stimulated relative to thecontralateral, buffer-injected joint FIG. 16). Thus, even underconditions in which insulin could be cleared from the synovial fluidand/or taken up by surrounding tissues and cells, articular cartilagehas an anabolic response to the insulin released by the PLGA-Insmicrospheres.

Taken together, our results clearly demonstrate that insulin mitigatedloss of matrix molecules such as proteoglycans as well as stimulatedsynthesis of new molecules to replace those lost. The net effect was toincrease the total amount of proteoglycans within a given amount ofcartilage tissue. This effect of insulin was shown in vitro and in vivo.Finally, we have created a slow-release formulation of insulin usingPLGA as a carrier. These PLGA-Ins microspheres appear to have superiorstability and activity relative to that of insulin alone.

Example 9 Human Cartilage Explants

It has been well established that the GH/IGF/IGFBP system is involved inthe regulation of anabolic and metabolic homeostasis and that defects inthis system may adversely affect growth, physiology, and glycemiccontrol (Jones et al., Endocr. Rev., 16: 3-34 (1995); Davidson, Endocr.Rev., 8: 115-131 (1987); Moses, Curr. Opin. Endo. Diab., 4: 16-25(1997)).

IGF-1 has been proposed for the treatment or prevention ofosteoarthritis, because of its ability to stimulate both matrixsynthesis and cell proliferation in culture (Osborn, J. Orthop. Res., 7:35-42 (1989)). IGF-1 has been administered with sodium pentosanpolysulfate (PPS) (a chondrocyte catabolic activity inhibitor) toseverely osteoarthritic canines with the effect of reducing the severityof the disease by lowering the levels of active neutralmetalloproteinase in the cartilage. In the model of mildlyosteoarthritic canines, therapeutic intervention with IGF-1 and PPStogether appeared to successfully maintain cartilage structure andbiochemistry, while IGF alone was ineffective, as described inRogachefsky, Osteoarthritis and Cartilage, 1: 105-114 (1993);Rogachefsky et al., Ann. NY Acad. Sci., 732: 889-895 (1994). The use ofIGF-1 either alone or as an adjuvant with other growth factors tostimulate cartilage regeneration has been described in WO 91/19510, WO92/13565, U.S. Pat. No. 5,444,047, and EP 434,652.

IGF-1 has also been found useful in the treatment of osteoporosis inmammals exhibiting decrease bone mineral density and those exposed todrugs or environmental conditions that result in bone density reductionand potentially osteoporosis, as described in EP 560,723 and EP 436,469.

IGF-1 insufficiency may have an etiologic role in the development ofosteoarthritis (Coutts et al., “Effect of growth factors on cartilagerepair,” Instructional Course Lect., 47: 487-494 (Amer. Acad. Orthop.Surg.: Rosemont, Ill. 1997)). Some studies indicate that serum IGF-1concentrations are lower in osteoarthritc patients than control groups,while other studies have found no difference. Nevertheless, it has beenshown that both serum IGF-1 levels and chrondrocyte responsiveness toIGF-1 decrease with age, with the latter likely due to high levels ofIGF-BPs (Florini and Roberts, J. Gerontol., 35: 23-30 (1980); Martin etal., J. Orthop. Res., 15: 491-498 (1997); Fernihough et al., Arthr.Rheum. 39: 1556-1565 (1996)). Thus, both the decreased availability ofIGF-1 as well as diminished chondrocyte responsiveness/disregulation ofIGFBPs thereto may contribute to the impaired cartilage matrixhomeostasis and tissue degeneration that occurs with advancing age anddisease.

The biological function of IGF binding proteins (IGFBPs) is not known.Of the IGFBPs, IGFBP-3 appears to be the IGFBP most responsible forregulating the total levels of IGF-1 and IGF-2 in plasma. IGFBP-3 is aGH-dependent protein and is reduced in cases of GH-deficiency orresistance (Jones et al., supra; Rosenfield et al., “IGF-1 treatment ofsyndromes of growth hormone insensitivity” In: The insulin-like growthfactors and their regulatory proteins, Eds Baxter R C, Gluckman P D,Rosenfield R G. Excerpta Medica, Amsterdam, 1994), pp 357-464; Scharf etal., J. Hepatology, 25: 689-699 (1996)). IGFBPs are able to enhance ofinhibit IGF activity, depending largely on their post-translationalmodifications and tissue localization (reviewed in Jones and Clemmons,Endocr. Rev. 16:3-34 (1995); Collet-Solberg and Cohen, Endocrinol.Metabol. Clin. North Am. 25:591-614 (1996)). In addition, disregulationin IGFBPs (3,4 and/or 5) may play a key role in arthritic disorders(Chevalier and Tyler, Brit. J. Rheum. 35: 515-522 (1996); Olney et al.,J. Clin. Endocrinol. Metab. 81: 1096-1103 (1996); Martel-Pelletier etal., Inflamm. Res., 47: 90-100 (1998)). It has been reported that IGF-1analogs with very low binding affinity for IGF-BPs were more effectivethan wild-type IGF-1 in stimulating proteoglycan synthesis (Morales,Arch Biochem. Biophys. 324, 173-188 (1997)). More recent data, however,suggest that IGFBPs contribute to IGF binding to and transport throughcartilage tissue, and IGFBPs may thus regulate bioavailability of IGF-1within the joint (Bhatka et al. J. Biol. Chem. 275: 5860-5866 (2000)).

WO 94/04569 discloses a specific binding molecule, other than a naturalIGFBP, that is capable of binding to IGF-1 and can enhance thebiological activity of IGF-1. WO 98/45427 published Oct. 15, 1998;Lowman et al., Biochemistry, 37: 8870-8878 (1998); and Dubaquie andLowman, Biochemistry, 38: 6386 (1999) disclose IGF-1 agonists identifiedby phage display. Also, WO 97/39032 discloses ligand inhibitors ofIGFBP's and methods for their use. Further, U.S. Pat. No. 5,891,722discloses antibodies having binding affinity for free IGFBP-1 anddevices and methods for detecting free IGFBP-1 and a rupture in a fetalmembrane based on the presence of amniotic fluid in a vaginal secretion,as indicated by the presence of free IGFBP-1 in the vaginal secretion.

Results and Discussion:

IGF-1 is a key regulator of matrix homeostasis in articular cartilage.The metabolic imbalance in osteoarthritis that favors matrix breakdownover new matrix synthesis may be due, at least in part, to insensitivityof chondrocytes to IGF-1 stimulation. While the mechanism underlyingthis IGF-1 resistance is not known, without being limited to any onetheory, it is believed that IGF-binding proteins (IGFBPs), which areelevated in many OA patients, play a role. In these patients, proteinswith IGF-1-like activity, which do not bind to, and are thus notinhibited by, IGFBPs would likely stimulate cartilage repair in tissuethat is otherwise IGF-1 resistant. As exemplied by FIG. 19, we havefound that at least 20% of patients undergoing joint replacement havearticular cartilage which is not responsive to IGF-I. However, thecartilage of these patients does respond to insulin, which does not bindto IGFBPs, by inducing significant synthesis of new cartilage matrixmolecules (FIGS. 19A,B). The fact that an IGF-I mutant, desIGF, whichdoes not bind to IGFBPs, induces cartilage matrix synthesis in theseIGF-I resistant tissues (FIGS. 19A,B), suggests that the mechanismunderlying IGF-1 resistance in this system is increased production ofinhibitory IGF-BPs. Thus, human, diseased cartilage remains responsiveto the anabolic effects of insulin, and as such, insulin is likely tohave beneficial effects on cartilage within diseased joints. Inaddition, insulin is expected to have anabolic effects on tissues, suchas arthritic cartilage, which are otherwise IGF-1 resistant.

Example 10 Expression of Insulin or Insulin Variants in E. coli

This example illustrates preparation of an unglycosylated form ofinsulin or insulin variants by recombinant expression in E. coli.

The DNA sequence encoding the insulin or insulin variant is initiallyamplified using selected PCR primers. The primers should containrestriction enzyme sites which correspond to the restriction enzymesites on the selected expression vector. A variety of expression vectorsmay be employed. An example of a suitable vector is pBR322 (derived fromE. coli; see Bolivar et al., Gene, 2:95 (1977)) which contains genes forampicillin and tetracycline resistance. The vector is digested withrestriction enzyme and dephosphorylated. The PCR amplified sequences arethen ligated into the vector. The vector will preferably includesequences which encode for an antibiotic resistance gene, a trppromoter, a polyhis leader (including the first six STII codons, polyhissequence, and enterokinase cleavage site), the insulin/insulin variantcoding region, lambda transcriptional terminator, and an argu gene.

The ligation mixture is then used to transform a selected E. coli strainusing the methods described in Sambrook et al., supra. Transformants areidentified by their ability to grow on LB plates and antibioticresistant colonies are then selected. Plasmid DNA can be isolated andconfirmed by restriction analysis and DNA sequencing.

Selected clones can be grown overnight in liquid culture medium such asLB broth supplemented with antibiotics. The overnight culture maysubsequently be used to inoculate a larger scale culture. The cells arethen grown to a desired optical density, during which the expressionpromoter is turned on.

After culturing the cells for several more hours, the cells can beharvested by centrifugation. The cell pellet obtained by thecentrifugation can be solubilized using various agents known in the art,and the solubilized insulin or insulin variant protein can then bepurified using a metal chelating column under conditions that allowtight binding of the protein.

Insulin or insulin variant may also be expressed in E. coli in apoly-His tagged form, using the following procedure. The DNA encodinginsulin or insulin variant is initially amplified using selected PCRprimers. The primers contain restriction enzyme sites which correspondto the restriction enzyme sites on the selected expression vector, andother useful sequences providing for efficient and reliable translationinitiation, rapid purification on a metal chelation column, andproteolytic removal with enterokinase. The PCR-amplified, poly-Histagged sequences are then ligated into an expression vector, which isused to transform an E. coli host based on strain 52 (W3110 fuhA(tonA)lon galE rpoHts(htpRts) clpP(lacIq). Transformants are first grown in LBcontaining 50 mg/ml carbenicillin at 30° C. with shaking until anO.D.600 of 3-5 is reached. Cultures are then diluted 50-100 fold intoCRAP media (prepared by mixing 3.57 g (NH₄)₂SO₄, 0.71 g sodium citrate2H₂ O, 1.07 g KCl, 5.36 g Difco yeast extract, 5.36 g Sheffield hycaseSF in 500 mL water, as well as 110 mM MPOS, pH 7.3, 0.55% (w/v) glucoseand 7 mM MgSO₄) and grown for approximately 20-30 hours at 30° C. withshaking. Samples are removed to verify expression by SDS-PAGE analysis,and the bulk culture is centrifuged to pellet the cells. Cell pelletsare frozen until purification and refolding.

E. coli paste from 0.5 to 1 L fermentations (6-10 g pellets) isresuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris, pH 8buffer. Solid sodium sulfite and sodium tetrathionate is added to makefinal concentrations of 0.1M and 0.02 M, respectively, and the solutionis stirred overnight at 4° C. This step results in a denatured proteinwith all cysteine residues blocked by sulfitolization. The solution iscentrifuged at 40,000 rpm in a Beckman Ultracentifuge for 30 min. Thesupernatant is diluted with 3-5 volumes of metal chelate column buffer(6 M guanidine, 20 mM Tris, pH 7.4) and filtered through 0.22 micronfilters to clarify. Depending on condition, the clarified extract isloaded onto a 5 ml Qiagen Ni-NTA metal chelate column equilibrated inthe metal chelate column buffer. The column is washed with additionalbuffer containing 50 mM imidazole (Calbiochem, Utrol grade), pH 7.4. Theprotein is eluted with buffer containing 250 mM imidazole. Fractionscontaining the desired protein were pooled and stored at 4° C. Proteinconcentration is estimated by its absorbance at 280 nm using thecalculated extinction coefficient based on its amino acid sequence.

The proteins are refolded by diluting sample slowly into freshlyprepared refolding buffer consisting of: 20 mM Tris, pH 8.6, 0.3 M NaCl,2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM EDTA. Refoldingvolumes are chosen so that the final protein concentration is between 50to 100 micrograms/ml. The refolding solution is stirred gently at 4° C.for 12-36 hours. The refolding reaction is quenched by the addition ofTFA to a final concentration of 0.4% (pH of approximately 3). Beforefurther purification of the protein, the solution is filtered through a0.22 micron filter and acetonitrile is added to 2-10% finalconcentration. The refolded protein is chromatographed on a Poros R1/Hreversed phase column using a mobile buffer of 0.1% TFA with elutionwith a gradient of acetonitrile from 10 to 80%. Aliquots of fractionswith A280 absorbance are analyzed on SDS polyacrylamide gels andfractions containing homogeneous refolded protein are pooled. Generally,the properly refolded species of most proteins are eluted at the lowestconcentrations of acetonitrile since those species are the most compactwith their hydrophobic interiors shielded from interaction with thereversed phase resin. Aggregated species are usually eluted at higheracetonitrile concentrations. In addition to resolving misfolded forms ofproteins from the desired form, the reversed phase step also removesendotoxin from the samples.

Fractions containing the desired folded insulin or insulin variantproteins, respectively, are pooled and the acetonitrile removed using agentle stream of nitrogen directed at the solution. Proteins areformulated into 20 mM Hepes, pH 6.8 with 0.14 M sodium chloride and 4%mannitol by dialysis or by gel filtration using G25 Superfine(Pharmacia) resins equilibrated in the formulation buffer and sterilefiltered.

Example 11 Expression of Insulin or Insulin Variants in Mammalian Cells

This example illustrates preparation of a potentially glycosylated formof insulin or insulin variants by recombinant expression in mammaliancells.

The vector, pRK5 (see EP 307,247, published Mar. 15, 1989), is employedas the expression vector. Optionally, the insulin or insulin variant DNAis ligated into pRK5 with selected restriction enzymes to allowinsertion of the insulin or insulin variant DNA using ligation methodssuch as described in Sambrook et al., supra. The resulting vector iscalled, for example, pRK5-ins.

In one embodiment, the selected host cells may be 293 cells. Human 293cells (ATCC CCL 1573) are grown to confluence in tissue culture platesin medium such as DMEM supplemented with fetal calf serum andoptionally, nutrient components and/or antibiotics. About 10 μg pRK5-insDNA is mixed with about 1 μg DNA encoding the VA RNA gene [Thimmappayaet al., Cell, 31:543 (1982)] and dissolved in 500 μL of 1 mM Tris-HCl,0.1 mM EDTA, 0.227 M CaCl₂. To this mixture is added, dropwise, 500 μLof 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaPO₄, and a precipitateis allowed to form for 10 minutes at 25° C. The precipitate is suspendedand added to the 293 cells and allowed to settle for about four hours at37° C. The culture medium is aspirated off and 2 ml of 20% glycerol inPBS is added for 30 seconds. The 293 cells are then washed with serumfree medium, fresh medium is added and the cells are incubated for about5 days.

Approximately 24 hours after the transfections, the culture medium isremoved and replaced with culture medium (alone) or culture mediumcontaining 200 uCi/ml ³⁵S-cysteine and 200 μCi/ml ³⁵S-methionine. Aftera 12 hour incubation, the conditioned medium is collected, concentratedon a spin filter, and loaded onto a 15% SDS gel. The processed gel maybe dried and exposed to film for a selected period of time to reveal thepresence of insulin or insulin variant polypeptide. The culturescontaining transfected cells may undergo further incubation (in serumfree medium) and the medium is tested in selected bioassays.

In an alternative technique, insulin or insulin variant may beintroduced into 293 cells transiently using the dextran sulfate methoddescribed by Somparyrac et al., Proc. Natl. Acad. Sci., 12:7575 (1981).293 cells are grown to maximal density in a spinner flask and 700 μgpRK5-ins DNA is added. The cells are first concentrated from the spinnerflask by centrifugation and washed with PBS. The DNA-dextran precipitateis incubated on the cell pellet for four hours. The cells are treatedwith 20% glycerol for 90 seconds, washed with tissue culture medium, andre-introduced into the spinner flask containing tissue culture medium, 5μg/ml bovine insulin and 0.1 μg/ml bovine transferrin. After about fourdays, the conditioned media is centrifuged and filtered to remove cellsand debris. The sample containing expressed insulin or insulin variantcan then be concentrated and purified by any selected method, such asdialysis and/or column chromatography.

In another embodiment, insulin or insulin variant can be expressed inCHO cells. The pRK5-ins can be transfected into CHO cells using knownreagents such as CaPO₄ or DEAE-dextran. As described above, the cellcultures can be incubated, and the medium replaced with culture medium(alone) or medium containing a radiolabel such as ³⁵S-methionine. Afterdetermining the presence of insulin or insulin variant, the culturemedium may be replaced with serum free medium. Preferably, the culturesare incubated for about 6 days, and then the conditioned medium isharvested. The medium containing the expressed insulin or insulinvariant can then be concentrated and purified by any selected method.

Epitope-tagged insulin or insulin variant may also be expressed in hostCHO cells. The insulin or insulin variant may be subcloned out of thepRK5 vector. The subclone insert can undergo PCR to fuse in frame with aselected epitope tag such as a poly-his tag into a Baculovirusexpression vector. The poly-his tagged insulin or insulin variant insertcan then be subcloned into a SV40 driven vector containing a selectionmarker such as DHFR for selection of stable clones. Finally, the CHOcells can be transfected (as described above) with the SV40 drivenvector. Labeling may be performed, as described above, to verifyexpression. The culture medium containing the expressed poly-His taggedinsulin or insulin variant can then be concentrated and purified by anyselected method, such as by Ni²⁺-chelate affinity chromatography.

Insulin or insulin variant may also be expressed in CHO and/or COS cellsby a transient expression procedure or in CHO cells by another stableexpression procedure.

Stable expression in CHO cells may be performed using the procedureoutlined below. The proteins may be expressed, for example, either (1)as an IgG construct (immunoadhesion), in which the coding sequences forthe soluble forms (e.g., extracellular domains) of the respectiveproteins are fused to an IgG constant region sequence containing thehinge CH2 domain and/or (2) a poly-His tagged form.

Following PCR amplification, the respective DNAs are subcloned in a CHOexpression vector using standard techniques as described in Ausubel etal., Current Protocols of Molecular Biology, Unit 3.16, John Wiley andSons (1997). CHO expression vectors are constructed to have compatiblerestriction sites 5′ and 3′ of the DNA of interest to allow theconvenient shuttling of cDNAs. The vector used expression in CHO cellsis as described in Lucas et al., Nucl. Acids Res. 24:9 (1774-1779(1996), and uses the SV40 early promoter/enhancer to drive expression ofthe cDNA of interest and dihydrofolate reductase (DHFR). DHFR expressionpermits selection for stable maintenance of the plasmid followingtransfection.

Twelve micrograms of the desired plasmid DNA is introduced intoapproximately 10 million CHO cells using commercially availabletransfection reagents Superfect® (Quiagen), Dosper® or Fugene®(Boehringer Mannheim). The cells are grown as described in Lucas et al.,supra. Approximately 3×10⁻⁷ cells are frozen in an ampule for furthergrowth and production as described below.

The ampules containing the plasmid DNA are thawed by placement intowater bath and mixed by vortexing. The contents are pipetted into acentrifuge tube containing 10 mLs of media and centrifuged at 1000 rpmfor 5 minutes. The supernatant is aspirated and the cells areresuspended in 10 mL of selective media (0.2 μm filtered PS20 with 5%0.2 μm diafiltered fetal bovine serum). The cells are then aliquotedinto a 100 mL spinner containing 90 mL of selective media. After 1-2days, the cells are transferred into a 250 mL spinner filled with 150 mLselective growth medium and incubated at 37° C. After another 2-3 days,250 mL, 500 mL and 2000 mL spinners are seeded with 3×10⁵ cells/mL. Thecell media is exchanged with fresh media by centrifugation andresuspension in production medium. Although any suitable CHO media maybe employed, a production medium described in U.S. Pat. No. 5,122,469,issued Jun. 16, 1992 may actually be used. A 3L production spinner isseeded at 1.2×10⁶ cells/mL. On day 0, the cell number pH is determined.On day 1, the spinner is sampled and sparging with filtered air iscommenced. On day 2, the spinner is sampled, the temperature shifted to33° C., and 30 mL of 500 g/L glucose and 0.6 mL of 10% antifoam (e.g.,35% polydimethylsiloxane emulsion, Dow Corning 365 Medical GradeEmulsion) taken. Throughout the production, the pH is adjusted asnecessary to keep it at around 7.2. After 10 days, or until theviability dropped below 70%, the cell culture is harvested bycentrifugation and filtering through a 0.22 μm filter. The filtrate waseither stored at 4° C. or immediately loaded onto columns forpurification.

For the poly-His tagged constructs, the proteins are purified using aNi-NTA column (Qiagen). Before purification, imidazole is added to theconditioned media to a concentration of 5 mM. The conditioned media ispumped onto a 6 ml Ni-NTA column equilibrated in 20 mM Hepes, pH 7.4,buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5ml/min. at 4° C. After loading, the column is washed with additionalequilibration buffer and the protein eluted with equilibration buffercontaining 0.25 M imidazole. The highly purified protein is subsequentlydesalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl and4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column andstored at −80° C.

Immunoadhesin (Fc-containing) constructs are purified from theconditioned media as follows. The conditioned medium is pumped onto a 5ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Naphosphate buffer, pH 6.8. After loading, the column is washedextensively with equilibration buffer before elution with 100 mM citricacid, pH 3.5. The eluted protein is immediately neutralized bycollecting 1 ml fractions into tubes containing 275 μL of 1 M Trisbuffer, pH 9. The highly purified protein is subsequently desalted intostorage buffer as described above for the poly-His tagged proteins. Thehomogeneity is assessed by SDS polyacrylamide gels and by N-terminalamino acid sequencing by Edman degradation.

Example 12 Expression of Insulin or Insulin Variant in Yeast

The following method describes recombinant expression of insulin orinsulin variant in yeast.

First, yeast expression vectors are constructed for intracellularproduction or secretion of insulin or insulin variant from theADH2/GAPDH promoter. DNA encoding insulin or insulin variant and thepromoter is inserted into suitable restriction enzyme sites in theselected plasmid to direct intracellular expression of the insert. Forsecretion, DNA encoding the insert can be cloned into the selectedplasmid, together with DNA encoding the ADH2/GAPDH promoter, a nativesignal peptide or other heterologous mammalian signal peptide, or, forexample, a yeast alpha-factor or invertase secretory signal/leadersequence, and linker sequences (if needed) for expression of the insert.

Yeast cells, such as yeast strain AB110, can then be transformed withthe expression plasmids described above and cultured in selectedfermentation media. The transformed yeast supernatants can be analyzedby precipitation with 10% trichloroacetic acid and separation bySDS-PAGE, followed by staining of the gels with Coomassie Blue stain.

Recombinant insulin or insulin variant can subsequently be isolated andpurified by removing the yeast cells from the fermentation medium bycentrifugation and then concentrating the medium using selectedcartridge filters. The concentrate containing the crude polypeptide mayfurther be purified using selected column chromatography resins.

Example 13 Expression of Insulin or Insulin Variant inBaculovirus-Infected Insect Cells

The following method describes recombinant expression of insulin orinsulin variant in Baculovirus-infected insect cells.

The sequence coding for insulin or insulin variant is fused upstream ofan epitope tag contained within a baculovirus expression vector. Suchepitope tags include poly-his tags and immunoglobulin tags (like Fcregions of IgG). A variety of plasmids may be employed, includingplasmids derived from commercially available plasmids such as pVL1393(Novagen). Briefly, the sequence encoding insulin or insulin variant orthe desired portion of the coding sequence of this polypeptide [such asthe sequence encoding the extracellular domain of a transmembraneprotein or the sequence encoding the mature protein if the protein isextracellular] is amplified by PCR with primers complementary to the 5′and 3′ regions. The 5′ primer may incorporate flanking (selected)restriction enzyme sites. The product is then digested with thoseselected restriction enzymes and subcloned into the expression vector.

Recombinant baculovirus is generated by co-transfecting the aboveplasmid and BaculoGold® virus DNA (Pharmingen) into Spodopterafrugiperda (“Sf9”) cells (ATCC CRL 1711) using lipofectin (commerciallyavailable from GIBCO-BRL). After 4-5 days of incubation at 28° C., thereleased viruses are harvested and used for further amplifications.Viral infection and protein expression are performed as described byO'Reilley et al., Baculovirus expression vectors: A Laboratory Manual,Oxford: Oxford University Press (1994).

Expressed poly-his tagged insulin or insulin variant can then bepurified, for example, by Ni²⁺-chelate affinity chromatography asfollows. Extracts are prepared from recombinant virus-infected Sf9 cellsas described by Rupert et al., Nature, 362: 175-179 (1993). Briefly, Sf9cells are washed, resuspended in sonication buffer (25 mL Hepes, pH 7.9;12.5 mM MgCl₂; 0.1 mM EDTA; 10% glycerol; 0.1% NP-40; 0.4 M KCl), andsonicated twice for 20 seconds on ice. The sonicates are cleared bycentrifugation, and the supernatant is diluted 50-fold in loading buffer(50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 7.8) and filteredthrough a 0.45 μm filter. A Ni²⁺-NTA agarose column (commerciallyavailable from Qiagen) is prepared with a bed volume of 5 mL, washedwith 25 mL of water and equilibrated with 25 mL of loading buffer. Thefiltered cell extract is loaded onto the column at 0.5 mL per minute.The column is washed to baseline A₂₈₀ with loading buffer, at whichpoint fraction collection is started. Next, the column is washed with asecondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% glycerol, pH6.0), which elutes nonspecifically bound protein. After reaching A₂₈₀baseline again, the column is developed with a 0 to 500 mM imidazolegradient in the secondary wash buffer. One mL fractions are collectedand analyzed by SDS-PAGE and silver staining or Western blot withNi²⁺-NTA-conjugated to alkaline phosphatase (Qiagen). Fractionscontaining the eluted His₁₀-tagged insulin or insulin variant are pooledand dialyzed against loading buffer.

Alternatively, purification of the IgG tagged (or Fc tagged) insulin orinsulin variant can be performed using known chromatography techniques,including for instance, Protein A or Protein G column chromatography.

Alternatively still, the insulin or insulin variant molecules of theinvention may be expressed using a modified baculovirus procedureemploying Hi-5 cells. In this procedure, the DNA encoding the desiredsequence was amplified with suitable systems, such as Pfu (Stratagene),or fused upstream (5′-of) an epitope tag contained within a baculovirusexpression vector. Such epitope tags include poly-His tags andimmunoglobulin tags (like Fc regions of IgG). A variety of plasmids maybe employed, including plasmids derived from commercially availableplasmids such as pIE-1 (Novagen). The pIE1-1 and pIE1-2 vectors aredesigned for constitutive expression of recombinant proteins from thebaculovirus ie1 promoter in stably transformed insect cells. Theplasmids differ only in the orientation of the multiple cloning sitesand contain all promoter sequences known to be important forie1-mediated gene expression in uninfected insect cells as well as thehr5 enhancer element. pIE1-1 and pIE1-2 include the ie1translationinitiation site and can be used to produce fusion proteins. Briefly, thedesired sequence or the desired portion of the sequence (such as thesequence encoding the extracellular domain of the transmembrane protein)is amplified by PCR with primers complementary to the 5′ and 3′ regions.The 5′ primer may incorporate flanking (selected) restriction enzymesites. The product was then digested with those selected restrictionenzymes and subcloned into the expression vector. For example,derivatives of pIE1-1 can include the Fc region of human IgG (pb.PH.IgG)or an 8 histidine (pb.PH.His) tag downstream (3′-of) the desiredsequence. Preferably, the vector construct is sequenced forconfirmation.

Hi5 cells are grown to a confluency of 50% under the conditions of 27°C., no CO₂, no pen/strep. For each 150 mm plate, 30 μg of pIE basedvector containing the sequence was mixed with 1 ml Ex-Cell medium(Media: Ex-Cell 401+1/100 L-Glu JRH Biosciences #14401-78P (note: thismedia is light sensitive)). Separately, 100 μl of Cell Fectin(CellFECTIN, Gibco BRL +10362-010, pre-vortexed) is mixed with 1 ml ofEx-Cell medium. The two solutions are combined and incubated at roomtemperature for 15 minutes. 8 ml of Ex-Cell media is added to the 2 mlof DNA/CellFECTIN mix and this is layered on Hi5 cells that have beenwashed once with Ex-Cell media. The plate is then incubated in darknessfor 1 hour at room temperature. The DNA/CellFECTIN mix is thenaspirated, and the cells are washed once with Ex-Cell to remove excessCell FECTIN. 30 ml of fresh Ex-Cell media is added and the cells areincubated for 3 days at 28° C. The supernatant is harvested and theexpression of the sequence in the baculovirus expression vector isdetermined by batch binding of 1 ml of supernatant to 25 ml of Ni-NTAbeads (QIAGEN) for histidine tagged proteins of Protein-A SepharoseCL-4B beads (Pharmacia) for IgG tagged proteins followed by SDS-PAGEanalysis comparing to a known concentration of protein standard byCoomassie blue staining.

The conditioned media from the transfected cells (0.5 to 3 L) washarvested by centrifugation to remove the cells and filtered through0.22 micron filters. For the poly-His tagged constructs, the proteincomprising the sequence is purified using a Ni-NTA column (Qiagen).Before purification, imidazole at a flow rate of 4-5 ml/min. at 48° C.After loading, the column is washed with additional equilibrium bufferand the protein eluted with equilibrium buffer containing 0.25Mimidazole. The highly purified protein was then subsequently desaltedinto a storage buffer containing 10 mM Hepes, 0.14 M NaCl and 4%mannitol, pH 6.8 with a 25 ml G25 Superfine (Pharmacia) column andstored at −80° C.

Immunoadhesion (Fc-containing) constructs may also be purified from theconditioned media as follows: The conditioned media is pumped onto a 5ml Protein A column (Pharmacia) which had been previously equilibratedin 20 mM sodium phosphate buffer, pH 6.8. After loading, the column iswashed extensively with equilibrium buffer before elution with 100 mMcitric acid, pH 3.5. The eluted protein is immediately neutralized bycollecting 1 ml fractions into tubes containing 275 μl of 1 M Trisbuffer, pH 9. The highly purified protein is subsequently desalted intostorage buffer as described above for the poly-His tagged proteins. Thehomogeneity is assessed by SDS polyacrylamide gels and by N-terminalamino acid sequencing by Edman degradation.

Example 14 Preparation of Antibodies that Bind Insulin or InsulinVariants

This example illustrates preparation of monoclonal antibodies which canspecifically bind insulin or insulin variants.

Techniques for producing the monoclonal antibodies are known in the artand are described, for instance, in Goding, supra. Immunogens that maybe employed include purified insulin or insulin variants, fusionproteins containing insulin or insulin variants, and cells expressingrecombinant insulin or insulin variant on the cell surface. Selection ofthe immunogen can be made by the skilled artisan without undueexperimentation.

Mice, such as Balb/c, are immunized with the insulin or insulin variantimmunogen emulsified in complete Freund's adjuvant and injectedsubcutaneously or intraperitoneally in an amount from 1-100 micrograms.Alternatively, the immunogen is emulsified in MPL-TDM adjuvant (RibiImmunochemical Research, Hamilton, Mont.) and injected into the animal'shind foot pads. The immunized mice are then boosted 10 to 12 days laterwith additional immunogen emulsified in the selected adjuvant.Thereafter, for several weeks, the mice may also be boosted withadditional immunization injections. Serum samples may be periodicallyobtained from the mice by retro-orbital bleeding for testing in ELISAassays to detect anti-insulin or anti-insulin variant antibodies.

After a suitable antibody titer has been detected, the animals“positive” for antibodies can be injected with a final intravenousinjection of insulin or insulin variant. Three to four days later, themice are sacrificed and the spleen cells are harvested. The spleen cellsare then fused (using 35% polyethylene glycol) to a selected murinemyeloma cell line such as P3X63AgU1, available from ATCC, No. CRL 1597.

The fusions generate hybridoma cells which can then be plated in 96 welltissue culture plates containing HAT (hypoxanthine, aminopterin, andthymidine) medium to inhibit proliferation of non-fused cells, myelomahybrids, and spleen cell hybrids.

The hybridoma cells are screened in an ELISA for reactivity againstinsulin or insulin variant. Determination of “positive” hybridoma cellssecreting the desired monoclonal antibodies against insulin or insulinvariant is within the skill in the art.

The positive hybridoma cells can be injected intraperitoneally intosyngeneic Balb/c mice to produce ascites containing the anti-insulin oranti-insulin variant monoclonal antibodies. Alternatively, the hybridomacells can be grown in tissue culture flasks or roller bottles.Purification of the monoclonal antibodies produced in the ascites can beaccomplished using ammonium sulfate precipitation, followed by gelexclusion chromatography. Alternatively, affinity chromatography basedupon binding of antibody to protein A or protein G can be employed.

Example 15 Purification of Insulin or Insulin Variant Polypeptides UsingSpecific Antibodies

Native or recombinant insulin or insulin variant polypeptides may bepurified by a variety of standard techniques in the art of proteinpurification. For example, pro-insulin or pro-insulin variantpolypeptide, mature insulin or insulin variant polypeptide, orpre-insulin/pre-insulin variant polypeptide can be purified byimmunoaffinity chromatography using antibodies specific for thepolypeptide of interest. In general, an immunoaffinity column isconstructed by covalently coupling the anti-insulin or anti-insulinvariant polypeptide antibody to an activated chromatographic resin.

Polyclonal immunoglobulins are prepared from immune sera either byprecipitation with ammonium sulfate or by purification on immobilizedProtein A (Pharmacia LKB Biotechnology, Piscataway, N.J.). Likewise,monoclonal antibodies are prepared form mouse ascites fluid by ammoniumsulfate precipitation or chromatography on immobilized Protein A.Partially purified immunoglobulin is covalently attached to achromatographic resin such as CnBr-activated SEPHAROSE™ (Pharmacia LKBBiotechnology). The antibody is coupled to the resin, the resin isblocked, and the derivative resin is washed according to themanufacturer's instructions.

Such an immunoaffinity column is utilized in the purification of insulinor insulin variant polypeptide by preparing a fraction from cellscontaining insulin or insulin variant polypeptide in a soluble form.This preparation is derived by solubilization of the whole cell or of asubcellular fraction obtained via differential centrifugation by theaddition of detergent or by other methods well known in the art.Alternatively, soluble insulin or insulin variant polypeptide containinga signal sequence may be secreted in useful quantity into the medium inwhich the cells are grown.

A soluble insulin or insulin variant polypeptide-containing preparationis passed over the immunoaffinity column, and the column is washed underconditions that allow the preferential absorbance of insulin or insulinvariant polypeptide (e.g., high ionic strength buffers in the presenceof detergent). Then, the column is eluted under conditions that disruptantibody/antigen binding (e.g., a low pH buffer such as approximately pH2-3, or a high concentration of a chaotrope such as urea or thiocyanateion), and insulin or insulin variant polypeptide is collected.

Example 16 Rational Drug Design

The goal of rational drug design is to produce structural analogs ofbiologically active polypeptide of interest (i.e., an insulin or insulinvariant polypeptide) or of small molecules with which they interact,e.g., agonists, antagonists, or inhibitors. Any of these examples can beused to fashion drugs which are more active or stable forms of theinsulin or insulin variant polypeptide or which enhance or interferewith the function of this polypeptide in vivo (c.f., Hodgson,Bio/Technology 9: 19-21 (1991)).

Insulin consists of an A (21 amino acids) and B (30 amino acids) chainwhich are linked through a pair of disulfide bonds. Across differentvertebrate species, minor differences in the primary structure ofinsulin exist (see diagram 1, reprinted from body [Hadley, M. E.Endocrinology, Prentice-Hall, Inc., 1988]. In mammals these differenceare usually in the 8, 9 and 10 positions within the intrachaindissulfide bond of the A chain and position 30 of the B chain. Althoughthese differences do not appear to affect biological potency, they canbe sufficient to make insulin antigenic in some patients [Hadley, M. E.,supra]. Insulin variants can be created which vary in time of action.For example, a rapid-acting form of insulin is created by reversingamino acids 28 and 29 on the insulin B-chain (Humalog, insulin lisproinjection, Eli Lilly). Alternatively, an intermediate-acting insulinwith a slower onset and a longer duration of activity (up to 24 hours)results when insulin is formulated as an amorphous and crystallinesuspension with zinc. (humulin L, Lente, Eli Lilly). The wealth ofinformation about the primary amino acid sequence of insulin as well asclinical data about immune reactions in humans to exogenous insulincould help in the design of insulin variants.

In one approach, the three-dimensional structure of the insulin orinsulin variant polypeptide, or of this polypeptide-inhibitor complex,is determined by x-ray crystallography, by computer modeling, or mosttypically, by a combination of these approaches. Both the shape andcharges of the insulin or insulin variant polypeptide must beascertained to elucidate the structure and to determine active site(s)of the molecule. Less often, useful information regarding the structureof the insulin or insulin variant polypeptide may be gained by modelingbased on the structure of homologous proteins. In both cases, relevantstructural information is used to design analogous insulin or insulinvariant polypeptide-like molecules or to identify efficient inhibitors.Useful examples of rational drug design may include molecules which haveimproved activity or stability as shown by Braxton and Wells,Biochemistry 31: 7796-7801 (1992) or which act as inhibitors, agonists,or antagonists of native peptides as shown by Athauda et al., J.Biochem. 113: 742-746 (1993).

It is also possible to isolate a target-specific antibody, selected byfunctional assay, as described above, and then to solve its crystalstructure. This approach, in principle, yields a pharmacore upon whichsubsequent drug design can be based. It is possible to bypass proteincrystallography altogether by generating anti-idiotypic antibodies(anti-ids) to a functional, pharmacologically active antibody. As amirror image of a mirror image, the binding site of the anti-ids wouldbe expected to be an analog of the original receptor. The anti-id couldthen be used to identify and isolate peptides from banks of chemicallyor biologically produced peptides. The isolated peptides would then actas the pharmacore.

By virtue of the present invention, sufficient amounts of the insulin orinsulin variant polypeptide may be made available to perform suchanalytical studies as X-ray crystallography. In addition, knowledge ofthe insulin or insulin variant polypeptide amino acid sequence providedherein will provide guidance to those employing computer modelingtechniques in place of or in addition to x-ray crystallography.

REFERENCES

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1. A method of treating damaged cartilage comprising contacting thecartilage by local administration through direct injection with insulinor insulin variant in an amount effective to (a) retain proteoglycans inthe matrix, (b) inhibit proteoglycan release from the matrix, or (c)stimulate proteoglycan synthesis.
 2. The method of claim 1, wherein thecartilage is articular cartilage.
 3. The method of claim 1, wherein thecartilage is contained in a mammal and the amount administered is atherapeutically effective amount.
 4. The method of claim 1, wherein thecartilage is damaged as a result of a degenerative disorder.
 5. Themethod of claim 4, wherein the degenerative cartilaginous disorder isrheumatoid arthritis.
 6. The method of claim 4, wherein the degenerativecartilaginous disorder is osteoarthritis.
 7. The method of claim 1,wherein the cartilage damage results from an injury.
 8. The method ofclaim 7, wherein the type of injury is a microdamage or blunt trauma, achondral fracture, an osteochondral fracture or damage to meniscus,tendon or ligament.
 9. The method of claim 7, wherein the injury is theresult of excessive mechanical stress or other biomechanical instabilityresulting from a sports injury or obesity.
 10. The method of claim 1,wherein the insulin or insulin variant is present in the form of acomposition further comprising a carrier, excipient or stabilizer. 11.The method of claim 10, wherein the cartilage is present in a mammal,and the amount administered is a therapeutically effective amount. 12.The method of claim 11, wherein the composition is an extended- orsustained-release formulation.
 13. The method of claim 12, wherein thecomposition further comprises poly-lactic-co-glycolic acid.
 14. Themethod of claim 12, wherein the composition further comprises apolyvalent metal salt.
 15. The method of claim 14, wherein thepolyvalent metal salt is zinc acetate.
 16. The method of claim 1,wherein the cartilage is present in a mammal and the amount administeredof insulin or insulin variant and cartilage agent is a therapeuticallyeffective amount.
 17. The method of claim 16, wherein the treatmentfurther comprises contacting the cartilage with a therapeuticallyeffective amount of a peptide growth factor selected from a familymember from the group consisting of: IGF (1,2), PDGF (AA, AB, BB), BMPs,FGF (1-20), TGF-β (1-3) and EGF.
 18. The method of claim 16, wherein thetreatment further comprises contacting the cartilage with atherapeutically effective amount of a catabolism antagonist selectedfrom the group consisting of IL-1ra, NO inhibitors, ICE inhibitor,agents which inhibit activity of IL-6, IL-8, LIF, IFN-γ, TNFαactivity,tetracyclines and variants thereof, inhibitors of apoptosis, MMPinhibitors, aggrecanase inhibitors and inhibitors of serine and cysteineproteinases (such as cathepsins and urokinase or tissue plasminogenactivator (uPA or tPA)).
 19. The method of claim 16, wherein thetreatment further comprises contacting the cartilage with atherapeutically effective amount of a osteo-factor selected from thegroup consisting of bisphosphonates, osteoprotegerin.
 20. The method ofclaim 16, wherein the comprises contacting the cartilage with atherapeutically effective amount of a anti-inflammatory factor isselected from the group consisting of anti-TNFα, soluble TNF receptors,IL1ra, soluble IL1 receptors, IL4, IL-10 and IL-13.
 21. The method ofclaim 1, wherein the treatment further comprises a standard surgicaltechnique selected from the group consisting of: cartilage shaving,abrasion chondroplasty, laser repair, debridement, chondroplasty,microfracture with or without subchondral bone penetration,mosaicplasty, cartilage cell allografts, stem cell autografts, coastalcartilage grafts, perichondral autografts, periosteal autografts,cartilage scaffolds, shell (osteoarticular) autografts or albografts andosteotomy.
 22. The method of claim 1, wherein the treatment furthercomprises chemical stimulation.
 23. The method of claim 1, wherein thetreatment further comprises electrical stimulation.
 24. A method ofreducing damage to cartilage in a patient with a cartilaginous disordercomprising contacting the cartilage by local administration throughdirect injection or extended release means with insulin or insulinvariant in an amount effective to (a) retain proteoglycans in thematrix, (b) inhibit proteoglycan release from the matrix, or (c)stimulate proteoglycan synthesis.
 25. The method of claim 24, whereinthe cartilage is articular cartilage.
 26. The method of claim 24,wherein the patient is a mammal and the amount administered is atherapeutically effective amount.
 27. The method of claim 26, whereinthe cartilage damage results from an injury.
 28. The method of claim 27,wherein the type of injury is a microdamage or blunt trauma, a chondralfracture, an osteochondral fracture or damage to meniscus, tendon orligament.
 29. The method of claim 27, wherein the injury is the resultof excessive mechanical stress or other biomechanical instabilityresulting from a sports injury or obesity.
 30. The method of claim 24,wherein the cartilage is damaged as a result of a degenerativecartilaginous disorder.
 31. The method of claim 30, wherein thedegenerative cartilaginous disorder is rheumatoid arthritis.
 32. Themethod of claim 30, wherein the degenerative cartilaginous disorder isosteoarthritis.
 33. The method of claim 24, wherein the insulin orinsulin variant is present in the form of a composition furthercomprising a carrier, excipient or stabilizer.
 34. The method of claim33, wherein the cartilage is present in a mammal, and the amountadministered is a therapeutically effective amount.
 35. The method ofclaim 33, wherein the composition is an extended- or sustained- releaseformulation.
 36. The method of claim 35, wherein the composition furthercomprises poly-lactic-co-glycolic acid.
 37. The method of claim 36,wherein the composition further comprises a polyvalent metal salt. 38.The method of claim 37, wherein the polyvalent metal salt is zincacetate.
 39. The method of claim 24, wherein the cartilage is present ina mammal and the amount administered of insulin or insulin variant andcartilage agent is a therapeutically effective amount.
 40. The method ofclaim 39, wherein the method further comprises contacting the cartilagewith an effective amount of a catabolism antagonist, selected from thegroup consisting of IL-1ra, NO inhibitors, ICE inhibitor, agents whichinhibit activity of IL-6, IL-8, LIF, IFN-γ, TNFα activity, tetracyclinesand variants thereof, inhibitors of apoptosis, MMP inhibitors,aggrecanase inhibitors and inhibitors of serine and cysteine proteinases(e.g. cathepsins, urokinase or tissue plasminogen activator (uPA ortPA)).
 41. The method of claim 39, wherein the method further comprisescontacting the cartilage with an effective amount of a peptide growthfactor selected from a family member from the group consisting of: IGF(1,2), PDGF (AA, AB, BB), BMPs, FGF (1-20), TGF-β (1-3) and EGF.
 42. Themethod of claim 39, wherein the method further comprises contacting thecartilage with an effective amount of a osteo-factor selected from thegroup consisting of bisphosphonates, osteoprotegerin.
 43. The method ofclaim 39, wherein the method further comprises contacting the cartilagewith an effective amount of an anti-inflammatory factor selected fromthe group consisting of anti-TNFα, soluble TNF receptors, IL1ra, solubleIL1 receptors, IL4, IL-10 and IL-13.